Method for efficient delivery of therapeutic molecules in vitro and in vivo

ABSTRACT

Compositions are described for direct protein delivery into multiple cell types in the mammalian inner ear. The compositions are used to deliver protein(s) (such as gene editing factors) editing of genetic mutations associated with deafness or associated disorders thereof. The delivery of genome editing proteins for gene editing and correction of genetic mutations protect or restore hearing from genetic deafness. Methods of treatment include the intracellular delivery of these molecules to a specific therapeutic target.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a national stage filing in accordance with 35 U.S.C. § 371 ofPCT/US2015/058092, filed Oct. 29, 2015, which claims the benefit of thepriority of U.S. Provisional Patent Application No. 62/072,209, filedOct. 29, 2014, the contents of each are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to protein therapeutics includinggenome-editing. Embodiments are directed to delivery of proteins thatare fused to an anionic molecule, antimicrobial or membranedestabilization molecules. These anionic molecules include, anoligonucleotide, a polynucleotide, negatively supercharged proteins,that contain natural anionic domains, or that natively bind to anionicnucleic acids.

BACKGROUND

Therapeutic proteins including peptide hormones, cytokines, andmonoclonal antibodies have achieved widespread success as research toolsand are among the fastest growing classes of drugs. Many powerful andpotentially therapeutic proteins have been discovered or engineered overthe past two decades, including enzymes capable of metaboliccomplementation (Hartung, S. D. et al. Gene. Mol. Ther. 9, 866-875(2004)), neutralizing antibodies against intracellular targets (Wang, J.et al. Nat. Biotechnol. 26, 901-908 (2008)), engineered transcriptionfactors (Urnov, F. D., et al. Nat. Rev. Genet. 11, 636-646 (2010)), andprogrammable genome-editing enzymes (Sander, J. D. & Joung, J. K. Nat.Biotechnol. 32, 347-355 (2014); Gaj, T., et al. Trends Biotechnol. 31,397-405 (2013)). While protein biologics have proven effective forextracellular targets, their use to address intracellular targets iscomparatively undeveloped due to the inability of most proteins tospontaneously enter mammalian cells. Enabling exogenous proteins toaccess intracellular targets is most commonly achieved by delivery oftheir encoding DNA sequences through chemical transfection (Midoux, P.,et al. Br. J. Pharmacol. 157, 166-178 (2009)), electroporation(Bodles-Brakhop, A. M., et al. Mol. Ther. 17, 585-592 (2009)), or viraldelivery (Kay, M. A., et al. Nat. Med. 7, 33-40 (2001)). Theintroduction of exogenous DNA into cells, however, raises thepossibility of permanent recombination into the genome, potentialdisruption of endogenous genes, and long-term exposure to the encodedagent. For some research or therapeutic applications, including genomeediting applications that seek to effect a one-time, permanentmodification of genomic DNA, the functional delivery of non-replicableprotein agents may offer improved safety or broader applicability.

The recent development of methods to deliver in vitro transcribed mRNAsor mRNA analogs has offered an alternative to DNA delivery withoutrequiring nuclear transport of an encoding gene, and with greatlyreduced potential for genomic insertion of the foreign nucleic acid.While promising, mRNA delivery continues to face challenges includingimmunogenicity and RNA stability. While chemical modifications and theinclusion of base analogs can mitigate some of these issues, thelarge-scale production of high-quality modified mRNAs remains achallenge (Zangi, L. et al. Nat. Biotechnol. 31, 898-907 (2013)).Moreover, proteins containing important natural or syntheticpost-translational modifications may not be amenable to production byendogenous translation machinery. Therefore, while both DNA and mRNAdelivery have become powerful research tools with therapeuticimplications, the development of effective and general protein deliverymethods remains an important challenge for the molecular life sciences.

Current or conventional protein delivery technologies are based onfusion or conjugation to cationic molecules that facilitate endocytosis,such as unstructured peptides (Wadia, J. S., et al. Nat. Med. 10,310-315 (2004); Daniels, D. S. & Schepartz, A. J. Am. Chem. Soc. 129,14578-14579 (2007)) or engineered superpositively charged proteins(Cronican, J. J. et al. ACS Chem. Biol. 5, 747-752 (2010); Thompson, D.B., et al. Methods Enzymol. 503, 293-319 (2012); Thompson, D. B., et al.Chem. Biol. 19, 831-843 (2012)). While such delivery can be effective incell culture, and has even shown some success in vivo, cationicprotein-based delivery methods have not seen widespread adoption.Unprotected proteins can be rapidly degraded by extracellular andendosomal proteases (Heitz, F., et al. Br. J. Pharmacol. 157, 195-206(2009)), or neutralized by binding to serum proteins, blood cells, andthe extracellular matrix (Caron, N. J. et al. Mol. Ther. J. Am. Soc.Gene Ther. 3, 310-318 (2001); Chesnoy, S. & Huang, L. Annu. Rev.Biophys. Biomol. Struct. 29, 27-47 (2000)). In addition, the lowefficiency of endosomal escape and avoidance of lysosomal degradationare major challenges to all endocytic protein delivery strategies, asevidenced by ongoing interest in endosome altering (Thompson, D. B., etal. Chem. Biol. 19, 831-843 (2012); Al-Taei, S. et al. Bioconjug. Chem.17, 90-100 (2006)) and destabilizing strategies (Shete, H. K., J.Nanosci. Nanotechnol. 14, 460-474 (2014)). These challenges have provenespecially difficult in vivo (Aguilera, T. A., et al. Integr. Biol.Quant. Biosci. Nano Macro 1, 371-381 (2009)).

Nucleic acid delivery has benefited greatly from the development ofliposomal reagents over the past two decades. Cationic lipidformulations have enabled DNA and RNA transfection to become a routinetechnique in basic research and have even been used in clinical trials(Coelho, T. et al. N. Engl. J. Med. 369, 819-829 (2013)). The lipidbilayer of the vehicle protects encapsulated nucleic acids fromdegradation and can prevent neutralization by antibodies (Judge, A. D.,et al. Mol. Ther. J. Am. Soc. Gene Ther. 13, 494-505 (2006)).Importantly, fusion of liposomes with the endosomal membrane duringendosome maturation can enable the efficient endosomal escape ofcationic lipid-delivered cargo (Basha, G. et al. Mol. Ther. J. Am. Soc.Gene Ther. 19, 2186-2200 (2011)). More advanced reversibly ionizablelipid nanoparticles enable efficient encapsulation and delivery ofnucleic acids, while avoiding non-specific electrostatic interactionsand sequestration (Semple, S. C. et al. Nat. Biotechnol. 28, 172-176(2010)).

Because proteins, in contrast to nucleic acids, are chemically diversewith no dominant electrostatic property, no lipid formulation is likelyto drive the efficient delivery of all proteins into mammalian cells.While proteins can be encapsulated non-specifically and delivered byrehydrated lipids in vitro (Boeckle, S., et al. J. Control. Release Off.J. Control. Release Soc. 112, 240-248 (2006); Allen, T. M. & Cullis, P.R. Adv. Drug Deliv. Rev. 65, 36-48 (2013)), the efficacy ofencapsulation is dependent on protein concentration, is generallyinefficient (Zelphati, O. et al. J. Biol. Chem. 276, 35103-35110(2001)), and has not seen widespread application. Specialty commercialreagents developed specifically for protein delivery (Adrian, J. E. etal. J. Control. Release Off. J. Control. Release Soc. 144, 341-349(2010); Morris, M. C., et al. Nat. Biotechnol. 19, 1173-1176 (2001))have also failed to garner popularity perhaps due to their low potencyand unreliability with a variety protein cargoes (Colletier, J.-P., etal. BMC Biotechnol. 2, 9 (2002)).

SUMMARY

Embodiments of the invention are directed to compositions comprisingtherapeutically effective anionically charged molecules and compositionsfor their efficient and specific delivery in vitro and in vivo. Targetedcells include any cells associated with hearing, including neurons, stemcells, hair cells, supporting cells and the like.

In an embodiment, a method of treating deafness or disorders thereof,associated with a genetic mutation in a patient in need thereof,comprises administering to an inner ear of the patient a therapeuticallyeffective amount of a chimeric molecule comprising at least one proteinor peptide fused, complexed or linked to one or more one or more geneediting agents, transcriptional modulators, translational modulators,post-translational modulators, and/or modulators that regulate proteinexpression, function, activity or combinations thereof. The chimericmolecule targets one or more genetic loci associated with deafness in apatient and modulates replication, expression, function or activity ofthe genetic locus in inner ear cells comprising, strial vascularis,neurons, hair cells, stem cells, or supporting cells. The chimericmolecule comprises a supercharged protein or variants thereof, forexample, the supercharged protein is green fluorescent protein (s-GFP),or variants thereof. The gene editing agents comprise: transcriptionalactivators, transcriptional repressors, recombinases, nucleases,DNA-binding proteins or nucleic acids, or combinations thereof. In someembodiments, the gene editing agents comprise: Cre recombinases,CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases,nickases, transcriptional regulators or combinations thereof.

In an embodiment, the supercharged protein further comprises a membranedestabilization protein, such, as for example, aurein, which is linkedto the s-GFP or variants thereof.

In some embodiments the chimeric molecule is encapsulated in a cationiclipid formulation.

In embodiments, the chimeric molecules target different chromosomalabnormalities, such as, for example, deletion, insertion, duplication,inversion and the like. The chimeric molecules are suitable therapeuticswhere monogenic, bigenic or multigenic mutations contribute to hearingloss. Accordingly, in other embodiments, the chimeric molecules targetmultiple genes.

In another embodiment, a method of correcting, silencing or modifying agenetic mutation associated with deafness or disorders thereof, in apatient, comprises administering to a patient's inner ear, a cationicliposome encapsulating a therapeutically effective amount of a moleculecomprising a supercharged protein or peptide fused, complexed or linkedto one or more one or more gene editing agents, transcriptionalmodulators, translational modulators, post-translational modulators,and/or modulators that regulate gene expression, protein expression,function, activity or combinations thereof. The molecule targets one ormore genetic loci having a mutation associated with deafness ordisorders thereof, in a patient, wherein the molecule corrects, silencesor modifies a genetic mutation inner ear cells comprising, strialvascularis, hair cells, stem cells, or supporting cells. For geneediting such as CRISPR, Cas9 (protein) is complexed with gRNA (guideRNA, nucleic acid) with/without a template by liposome. Such complex canthen be delivered to inner ear for gene editing. For the disruption ofmutation, only gRNA without template is needed; whereas for mutationrepair, both a gRNA and a template are needed; in addition to Cas9.

In another embodiment, a method of correcting, silencing or modifying agenetic mutation in hair cells, stem cells, and/or supporting cells invitro or in vivo, comprises contacting a hair cell or supporting cell oradministering to a patient's inner ear, a therapeutically effectiveamount of a molecule comprising a supercharged protein or peptide fused,complexed or linked to one or more one or more gene editing agents,transcriptional modulators, translational modulators, post-translationalmodulators, and/or modulators that regulate protein expression,function, activity or combinations thereof. The molecule targets one ormore genetic loci having a mutation associated with deafness ordisorders thereof, in a patient, wherein the molecule corrects, silencesor modifies a genetic mutation in hair cells and supporting cells of theinner ear.

In another embodiment, a method of delivering a therapeutic molecule tocells of an inner ear of a patient, comprises administering to the innerear of a patient a chimeric molecule comprising at least one protein orpeptide fused, complexed or linked to one or more anionic molecules. Inan embodiment, the cells of the inner ear comprise hair cells,supporting cells, or combinations thereof. In an embodiment, thechimeric molecule comprises one or more gene editing agents,transcriptional modulators, translational modulators, post-translationalmodulators, and/or modulators that regulate gene expression, proteinexpression, function, activity or combinations thereof. The gene editingagents comprise: transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In an embodiment, the anionic molecules comprise:oligonucleotides, polynucleotides, proteins, peptides, peptide nucleicacids (PNA), synthetic molecules or combinations thereof. Theoligonucleotides or polynucleotides comprise: ribonucleic acids (RNA),deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modifiedRNA or DNA sequences, complementary DNA (cDNA), guide RNA (gRNA), shortguide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequencescomprising one or more modified nucleobases or backbones, orcombinations thereof. In other embodiments, the chimeric moleculecomprises a supercharged protein or variants thereof. An example of asupercharged protein is a fluorescent protein, or variants thereof. Thechimeric molecule further comprises a membrane destabilizing protein,for example, aurein.

In some embodiments, the chimeric molecule is encapsulated in a cationiclipid formulation.

In some embodiments, a composition comprises a cationic lipidencapsulating one or more chimeric molecules comprising one or moreproteins or peptides fused, complexed or linked to one or more anionicmolecules. These one or more anionic molecules confer an overall netnegative charge to the chimeric molecule and comprise one or moreanionic domains or bind to an anionic nucleic acid domain. In someembodiments, the anionic molecules comprise: oligonucleotides,polynucleotides, proteins, peptides, peptide nucleic acids (PNA),synthetic molecules or combinations thereof. In some embodiments, theoligonucleotides or polynucleotides comprise: ribonucleic acids (RNA),deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modifiedRNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA),interference RNA, mRNA, nucleic acid sequences comprising one or moremodified nucleobases or backbones, or combinations thereof.

In embodiments, the one or more proteins or peptides are cationic,anionic or are neutrally charged. In some embodiments, the proteins orpeptides comprise: enzymes, hormones, chemotherapeutic agents,immunotherapeutic agents, gene editing agents, synthetic molecules orcombinations thereof. In some embodiments, the gene editing agentscomprise: transcriptional activators, transcriptional repressors,transcription factors, enhancer modulating molecules, recombinases,nucleases, nucleic acid binding-proteins, nucleic acidbinding-polynucleotides or oligonucleotides, DNA-binding proteins orDNA-binding nucleic acids, or combinations thereof.

In other embodiments, methods of treatment comprises administering atherapeutically effective amount of a cationic lipid encapsulating oneor more chimeric molecules comprising one or more proteins or peptidesfused, complexed or linked to one or more anionic molecules.

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B show that after microinjection with Cas9 protein and gRNAcomplexed with lipofectamine2000, OHCs labeled with Myo7a/DAPI withoutGFP were seen in the adult Atoh1-GFP transgenic cochlea, a demonstrationof genome editing. The efficiency is ˜15%. In control cochlea injectedwith saline, all cochlear OHCs were positive for GFP, a demonstration ofspecificity of Cas9:gRNA in genome editing in adult cochlea in vivo.

FIG. 2 is a schematic representation showing a design of gRNA forCas9:gRNA (SEQ ID NOS: 21-33) injection. Mutation 2765C→T was shown.Multiple gRNAs, some target the mutation and some target wildtype Pmca2gene were designed for injection with Cas9.

FIG. 3 is a series of graphs showing that Cas9:gRNA injection restoredhearing in the Obl heterozygous mice. In the uninjected inner ears,profound hearing loss was shown by the complete lack of ABR and DPOAEacross all frequencies at the highest sound pressure level (SPL) from 4weeks to three months. In the injected inner ear, significant hearingrecovery was achieved across most frequencies (except for 45.25 kHz) byABR as well as by DPOAE. In the middle frequencies 11.32 to 22.64 kHz,the recovery was over 50 dB, a demonstration of restoration of hearingto near normal levels. At 8 weeks and three months of age, hearingrecovery was largely maintained shown by ABR and DPOAE. Deterioration inhigh frequency was seen at later stages.

FIG. 4 are graphs showing that hearing recovery is gRNA specific. Anexample of a gRNA-2.5-mut with different sequence did not result inhearing improvement after injection with Cas9. gRNAs against wildtypePmca2 did not improve hearing either.

FIG. 5 shows the high-efficiency cleavage by specific gRNA-2.4-mut.Different concentrations of Cas9 protein and gRNAs were tested for DNAcleavage in vitro. Only gRNA-2.4-mut showed cleavage in mutant but notin wildtype Pmca2 genomic region at 125 nM. Whereas other gRNAs showedcleavages in both mutant and wildtype genomic DNA.

FIG. 6A, 6B show that Cas9:gRNA injection promotes outer hair cellsurvival.

FIG. 6A: Two months after injection, a majority of outer hair cells areseen in the base compared to a complete outer hair cell loss in theuninjected control ear. FIG. 6B: Cell count showed general preservationof outer hair cells across all frequencies after Cas9:gRNA injection twomonths later. In contrast to almost complete outer hair cell loss athigh-frequencies 32 and 45.25 kHz, outer hair cell number in theinjected inner ears was virtually intact.

FIGS. 7A, 7B is a color stain showing the preservation of ganglionneurites in the Cas9:gRNA injected cochlea. FIG. 7A: In the controluninjected inner ear of Pmca2^(Obl/+) mice, TuJ labeling of neurites islargely absent in the region between outer hair cells (OHCs) and innerhair cells (IHCs) (Bracket demarcates the region). FIG. 7B: In theinjected inner ear in contrast, preservation of neurites is seen by alarge number of TuJ⁺ fibers. Parv: parvalbumin that labels hair cells.

FIG. 8 is a series of graphs showing the hearing rescue by Cas9:gRNA inthe Bth heterozygous mice. 4 weeks after injection, significant hearingimprovement is observed in the middle frequencies of 11.32 to 32.64 kHzin the injected ears comparing to control uninjected inner ears. TheDPOAE is similar with the exception at 11.32 kHz whereas injected earsshowed elevated DPOAE threshold shift.

FIG. 9 is a graph showing that in four weeks after multiple injections,ABR in the Cas9/gRNA-Pmca2-2.4 injected inner ears show dramatic hearingrestoration at 16, 22.64, 32 and 45.24 kHz comparing to the uninjectedcontrol ears. Hearing restoration at 16, 22.64 and 32 kHz are improvedby 40 dB.

FIGS. 10A and 10B show the in vivo delivery of Cre recombinase to haircells in the mouse inner ear. FIG. 10A: The scala media (cochlear duct)of P0 floxP-tdTomato mice (n=4) were injected with 0.3 μL of 23 μM(−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5days, tdTomato expression indicative of Cre-mediated recombination wasvisualized using immunohistology. Red=tdTomato; green=Myo7a; white=Sox2;blue=DAPI. Yellow brackets indicate the outer hair cell (OHC) region.FIG. 10B: Ten days after (−30)GFP-Cre delivery, intact espin(Esp)-expressing stereocilia of tdTomato-positive outer hair cells werepresent (arrow), similar to stereocilia in control cochlea.Red=tdTomato; green=Esp, white=Sox2; blue=DAPI, Scale bars: 10 μm.

FIGS. 11A-11E show the high efficiency of protein delivery mediated byaurein in the inner ear in vivo. Injection of (+36)GFP-aurein-Cre withdifferent concentrations to Rosa-tdTf^(/f) mouse cochlea resulted in tdTlabeling in cochlear cell types with different efficiencies. FIG. 11A:At 5 μM, tdT labeling was mainly in some IHC and SC. FIG. 11B: At 22.5μM, tdT labeling was in most IHC and OHC, and in some SC and spiralganglion neurons (SPN). FIG. 11C: At 50 μM, tdT was seen in most IHC andOHC, as well as a large number of SC. IHC loss was seen under thecondition. FIG. 11D: In mice injected with 50 μM (+36)GFP-Cre, very fewIHC and HC were labeled with tdT. FIG. 11E: In Opti-MEM injected controlmice no tdT labeling was detected in any inner ear cells. Scale bars: 10μm.

FIG. 12 shows the high efficiency of aurein mediated protein delivery inthe mammalian inner ear. Comparison between different concentrations of(+36)GFP-aurein-Cre and control reveled that extremely high-efficientdelivery of functional protein (Cre) with the aurein at 22.5 and 50 μM,respectively.

FIG. 13A is a schematic representation showing an overview of proteindelivery in mammalian cells. Cationic macromolecules such as +36 GFPinteract with anionic sulfated proteoglycans on the cell surface and areendocytosed and sequestered in early endosomes. The early endosomes canacidify into late endosomes or lysosomes. Alternatively, early endosomesmay be trafficked back to the cell surface as part of themembrane-recycling pathway. To access the cytoplasm, an exogenouscationic protein must escape endosomes before it is degraded orexported. FIG. 13B is a schematic representation showing thesortase-mediated conjugation of peptides with +36 GFP-Cre recombinaseprior to screening. Sortase was used to conjugate synthetic peptidescontaining a C-terminal LPETGG (SEQ ID NO: 89) with expressed +36GFP-Cre containing an N-terminal GGG. The resulting peptide-LPETGGG-+36GFP-Cre fusion proteins (“LPETGGG” disclosed as SEQ ID NO: 96) have thesame chemical composition as expressed recombinant proteins, but aremore easily assembled.

FIG. 14 is a graph showing a primary screen for cytosolic delivery ofCre recombinase in BSR.LNL.tdTomato cells. Initial screen of 20peptide-(+36 GFP)-Cre conjugated proteins. Cytosolic Cre deliveryresults in recombination and tdTomato expression. The percentage oftdTomato positive cells was determined by fluorescence image analysis.250 nM+36 GFP-Cre was used as the no-peptide control (NP), and additionof 100 μM chloroquine was used as the positive control (+). Cells weretreated with 250 nM protein for 4 h in serum-free DMEM. Cells werewashed and supplanted with full DMEM and incubated for 48 h. Error barsrepresent the standard deviation of three independent biologicalreplicates.

FIGS. 15A, 15B are graphs showing the efficacy and toxicity ofrecombinant expression fusions of aurein 1.2 (“E”) and citropin 1.3(“U”). FIG. 15A shows that the cytosolic Cre delivery results inrecombination and tdTomato expression. The percentage of tdTomatopositive cells was determined by flow cytometry. Protein fusions weredelivered at 125 nM, 250 nM, 500 nM, and 1 μM. FIG. 15B shows thetoxicity of aurein 1.2 and citropin 1.3 as determined by CELLTITERGLO(Promega) assay. Protein fusions were delivered at 125 nM, 250 nM, 500nM, and 1 μM. The labeled concentration of +36 GFP-Cre was used as theno peptide control (NP), and addition of 100 μM chloroquine was used asthe positive control (+). Cells were treated with 250 nM protein for 4 hin serum-free media. Cells were washed and supplanted with full DMEM andincubated for 48 h. Error bars represent the standard deviation of threeindependent biological replicates.

FIGS. 16A, 16B are graphs showing the activity and cytotoxicity ofaurein 1.2 variants fused to +36 GFP-Cre (FIG. 16A). The percentage oftdTomato positive cells was determined by flow cytometry. (FIG. 16B)Toxicity as determined by CELLTITERGLO (Promega) assay. 250 nM+36GFP-Cre was used as the no peptide control (NP), and addition of 100 μMchloroquine was used as the positive control (+). Cells were treatedwith 250 nM protein for 4 h in serum-free DMEM. Cells were washed andsupplanted with full DMEM and incubated for 48 h.

FIGS. 17A-17D show the ability of +36 GFP and aurein 1.2-+36 GFPdexamethasone-conjugates to reach the cytosol and activate GRtranslocation. FIG. 17A shows images of HeLa cells expressing GR-mCherrytreated in the presence and absence of 1 μM dexamethasone (Dex)-proteinconjugates for 30 min at 37° C. FIG. 17B shows that nuclear-to-cytosolGR-mCherry fluorescence ratios (translocation ratios) of respectiveDex-protein conjugates determined using CELLPROFILER®. FIG. 17C showsthe GR-mCherry translocation ratios resulting from cells treated in thepresence and absence of +36 GFP^(Dex) and endocytic inhibitors. FIG. 17Dshows the GR-mCherry translocation ratios resulting from cells treatedin the presence and absence of aurein 1.2-+36 GFP^(Dex) and endocyticinhibitors. Statistical significance is measured by P-value.ns=P>0.05, * =P≤0.05, ** =P≤0.01, *** =P≤0.001. FIG. 17 discloses “Arg8”disclosed as SEQ ID NO: 102.

FIGS. 18A-18C show the results from the in vivo protein delivery of Crerecombinase into mouse neonatal cochleas. 0.4 μL of 50 μM+36 GFP-Cre oraurein 1.2-+36 GFP-Cre were injected into the scala media. FIG. 18A:Five days after injection, cochlea were harvested. Inner hair cells(IHC), outer hair cells (OHC) and supporting cells in the sensoryepithelium (SE) were imaged for the presence of tdTomato, which is onlyexpressed following Cre-mediated recombination. Hair cells were labeledwith antibodies against the hair-cell marker Myo7a. Gray/white=Myo7a,Red=tdTomato, Blue=DAPI. FIG. 18B: To evaluate cytotoxicity, the numberof outer hair cells and inner hair cells were measured by countingDAPI-stained cells. FIG. 18C: The percentage of tdTomato positive cells,reflecting successful delivery of functional Cre recombinase, wasdetermined by fluorescence imaging.

DETAILED DESCRIPTION

Embodiments of the invention are directed to compositions for theefficient intracellular delivery of proteins to the nucleus orcytoplasm. Conventional methods of protein delivery typically rely oncationic peptides or proteins to facilitate endocytosis, but suffer fromlow tolerance for serum proteins, poor endosomal escape, and limited invivo efficacy. Herein, it is reported that cationic lipid reagents canpotently deliver to hair cells and supporting cells, proteins that arefused to polynucleotides, oligonucleotides, negatively superchargedproteins, that contain natural anionic domains, or that natively bind toanionic nucleic acids.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes or geneproducts disclosed herein, which in some embodiments relate to mammaliannucleic acid and amino acid sequences, are intended to encompasshomologous and/or orthologous genes and gene products from other animalsincluding, but not limited to other mammals, fish, amphibians, reptiles,and birds. In preferred embodiments, the genes, nucleic acid sequences,amino acid sequences, peptides, polypeptides and proteins are human.

The following description of the preferred embodiments is merelyexemplary in nature and is in no way intended to limit the invention,its application or uses. Embodiments of the invention may be practicedwithout the theoretical aspects presented. Moreover, the theoreticalaspects are presented with the understanding that Applicants do not seekto be bound by the theory presented.

It should be understood that numerous specific details, relationships,and methods are set forth to provide a full understanding of theinvention. One having ordinary skill in the relevant art, however, willreadily recognize that the invention can be practiced without one ormore of the specific details or with other methods. The presentinvention is not limited by the illustrated ordering of acts or events,as some acts may occur in different orders and/or concurrently withother acts or events. Furthermore, not all illustrated acts or eventsare required to implement a methodology in accordance with the presentinvention.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, in reference to defined or described elements of anitem, composition, apparatus, method, process, system, etc. are meant tobe inclusive or open ended, permitting additional elements, therebyindicating that the defined or described item, composition, apparatus,method, process, system, etc. includes those specified elements—or, asappropriate, equivalents thereof—and that other elements can be includedand still fall within the scope/definition of the defined item,composition, apparatus, method, process, system, etc.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value or range.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude within 5-fold,and also within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

As used herein, a “chimeric” molecule is one which comprises one or moreunrelated types of components or contain two or more chemically distinctregions or domains which can be conjugated to each other, fused, linked,translated, attached via a linker, chemically synthesized, expressedfrom a nucleic acid sequence, etc. For example, an antimicrobial ormembrane destabilizing peptide and an unrelated peptide, a peptide and anucleic acid sequence, a peptide and a detectable label, unrelatedpeptide sequences, unrelated nucleic acid sequence and the like.

The term “anionic” molecule is one which comprises one or more “anionic”domains which confer an overall net anionic charge to the molecule.Accordingly, the chimeric molecule can be an anionic molecule.

A “supercharged” molecule is a molecule e.g. peptide, having a positiveor negative charge and when it is connected to another molecule confersan overall positive or negative charge to the entire molecule.

As used herein, a “membrane destabilizing domain” is one which disruptsa cellular membrane in vitro or in vivo, for example, aurein. The termencompasses proteins, peptides, polynucleotides, oligonucleotides,bacterial or viral molecules, antimicrobial peptides (AMP),antibacterial molecules, microtubules, synthetic or natural molecules. Achimeric molecule embodied herein, further comprises one or moremembrane destabilizing domains.

As used herein, the terms “associated with,” “conjugated,” “linked,”“attached,” and “tethered,” when used with respect to two or moremoieties, means that the moieties or domains are physically associatedor connected with one another, either directly or via one or moreadditional moieties that serves as a linking agent, to form a structurethat is sufficiently stable so that the moieties remain physicallyassociated under the conditions in which the structure is used, e.g.,physiological conditions. The term “connected” will be used for the sakeof brevity and is meant to include all possible methods of physicallyassociating each domain of the chimeric molecule to each other. Forexample, a supercharged protein is typically associated with orconnected to a nucleic acid by a mechanism that involves non-covalentbinding (e.g., electrostatic interactions). In certain embodiments, apositively charged, supercharged protein is associated with a nucleicacid through electrostatic interactions to form a complex. In someembodiments, a sufficient number of weaker interactions can providesufficient stability for moieties to remain physically associated undera variety of different conditions. In certain embodiments, the agent tobe delivered is covalently bound to the supercharged protein.

As used herein, the term “green fluorescent protein” (GFP) refers to aprotein originally isolated from the jellyfish Aequorea victoria thatfluoresces green when exposed to blue light or a derivative of such aprotein (e.g., a supercharged version of the protein). The amino acidsequence of wild type GFP is as follows:

(SEQ ID NO: 20) MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFICTT GKLPVPWPTL VTTFSYGVQC FSRYPDHMKQHDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLVNRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNGIKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHYLSTQSALSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK.

Proteins that are at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98% or at least 99% homologousare also considered to be green fluorescent proteins. In certainembodiments, the green fluorescent protein is supercharged. In certainembodiments, the green fluorescent protein is superpositively charged(e.g., +15 GFP, +25 GFP, and +36 GFP). In certain embodiments, the GFPmay be modified to include a polyhistidine tag for ease in purificationof the protein. In certain embodiments, the GFP may be fused withanother protein or peptide (e.g., hemagglutinin 2 (HA2) peptide). Incertain embodiments, the GFP may be further modified biologically orchemically (e.g., post-translational modifications, proteolysis, etc.).

As used herein, unless otherwise indicated, the terms “peptide”,“polypeptide” or “protein” are used interchangeably herein, and refer toa polymer of amino acids of varying sizes. These terms do not connote aspecific length of a polymer of amino acids. Thus, for example, theterms oligopeptide, protein, and enzyme are included within thedefinition of polypeptide or peptide, whether produced using recombinanttechniques, chemical or enzymatic synthesis, or be naturally occurring.This term also includes polypeptides that have been modified orderivatized, such as by glycosylation, acetylation, phosphorylation, andthe like.

As used herein, a “nucleic acid” or “nucleic acid sequence” or “cDNA”refers to a nucleic acid segment or fragment which has been separatedfrom sequences which flank it in a naturally occurring state, e.g., aDNA fragment which has been removed from the sequences which arenormally adjacent to the fragment, e.g., the sequences adjacent to thefragment in a genome in which it naturally occurs, and refers to nucleicacid sequences in which one or more introns have been removed. The termalso applies to nucleic acids which have been substantially purifiedfrom other components which naturally accompany the nucleic acid, e.g.,RNA or DNA or proteins, which naturally accompany it in the cell. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., as a cDNA or a genomic or cDNAfragment produced by PCR or restriction enzyme digestion) independent ofother sequences. It also includes a recombinant DNA, for instance, DNAwhich is part of a hybrid gene encoding additional polypeptidesequences.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

As used herein, the terms “nucleic acid sequence”, “polynucleotide,” and“gene” are used interchangeably throughout the specification and includecomplementary DNA (cDNA), linear or circular oligomers or polymers ofnatural and/or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, substituted and alpha-anomericforms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA),phosphorothioate, methylphosphonate, and the like.

The nucleic acid sequences may be “chimeric,” that is, composed ofdifferent regions. In the context of this invention “chimeric” compoundsare oligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide.These sequences typically comprise at least one region wherein thesequence is modified in order to exhibit one or more desired properties.

The term “target nucleic acid” refers to a nucleic acid (often derivedfrom a biological sample), to which the oligonucleotide is designed tospecifically hybridize. It is either the presence or absence of thetarget nucleic acid that is to be detected, or the amount of the targetnucleic acid that is to be quantified. The target nucleic acid has asequence that is complementary to the nucleic acid sequence of thecorresponding oligonucleotide directed to the target. The term targetnucleic acid may refer to the specific subsequence of a larger nucleicacid to which the oligonucleotide is directed or to the overall sequence(e.g., gene or mRNA) whose expression level it is desired to detect. Thedifference in usage will be apparent from context.

In the present context, the terms “nucleobase” covers naturallyoccurring nucleobases as well as non-naturally occurring nucleobases. Itshould be clear to the person skilled in the art that variousnucleobases which previously have been considered “non-naturallyoccurring” have subsequently been found in nature. Thus, “nucleobase”includes not only the known purine and pyrimidine heterocycles, but alsoheterocyclic analogues and tautomers thereof. Illustrative examples ofnucleobases are adenine, guanine, thymine, cytosine, uracil, purine,xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine,7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine,5-methylcytosine, 5-(C₃-C₆)-alkynylcytosine, 5-fluorouracil,5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin,isocytosine, isoguanin, inosine and the “non-naturally occurring”nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. Theterm “nucleobase” is intended to cover every and all of these examplesas well as analogues and tautomers thereof. Especially interestingnucleobases are adenine, guanine, thymine, cytosine, and uracil, whichare considered as the naturally occurring nucleobases in relation totherapeutic and diagnostic application in humans.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Kornberg andBaker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).

“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443,Toulmé, J. J., Nature Biotechnology 19:17-18 (2001); Manoharan M.,Biochemica et Biophysica Acta 1489:117-139(1999); Freier S. M., NucleicAcid Research, 25:4429-4443 (1997), Uhlman, E., Drug Discovery &Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic AcidDrug Dev., 10:297-310 (2000)); 2′-O, 3′-C-linked [3.2.0]bicycloarabinonucleosides (see e.g. N. K Christiensen., et al., J. Am.Chem. Soc., 120: 5458-5463 (1998). Such analogs include syntheticnucleosides designed to enhance binding properties, e.g., duplex ortriplex stability, specificity, or the like.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype target gene products. Variants may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orin polypeptides whose structure or function may or may not be altered.Any given natural or recombinant gene may have none, one, or manyallelic forms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

As used herein, “variant” of polypeptides refers to an amino acidsequence that is altered by one or more amino acid residues. The variantmay have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological activity may be foundusing computer programs well known in the art, for example, LASERGENEsoftware (DNASTAR).

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. “Treatment” may also bespecified as palliative care. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented. Accordingly, “treating” or “treatment” of a state,disorder or condition includes: (1) preventing or delaying theappearance of clinical symptoms of the state, disorder or conditiondeveloping in a human or other mammal that may be afflicted with orpredisposed to the state, disorder or condition but does not yetexperience or display clinical or subclinical symptoms of the state,disorder or condition; (2) inhibiting the state, disorder or condition,i.e., arresting, reducing or delaying the development of the disease ora relapse thereof (in case of maintenance treatment) or at least oneclinical or subclinical symptom thereof; or (3) relieving the disease,i.e., causing regression of the state, disorder or condition or at leastone of its clinical or subclinical symptoms. The benefit to anindividual to be treated is either statistically significant or at leastperceptible to the patient or to the physician.

The terms “patient” or “individual” or “subject” are usedinterchangeably herein, and refers to a mammalian subject to be treated,with human patients being preferred. In some cases, the methods of theinvention find use in experimental animals, in veterinary application,and in the development of animal models for disease, including, but notlimited to, rodents including mice, rats, and hamsters, and primates.

As defined herein, a “therapeutically effective” amount of a compound oragent (i.e., an effective dosage) means an amount sufficient to producea therapeutically (e.g., clinically) desirable result. The compositionscan be administered from one or more times per day to one or more timesper week; including once every other day. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof the compounds of the invention can include a single treatment or aseries of treatments.

As defined herein, an “effective” amount of a compound or agent (i.e.,an effective dosage) means an amount sufficient to produce a (e.g.,clinically) desirable result.

As used herein, a “pharmaceutically acceptable” component/carrier etc.is one that is suitable for use with humans and/or animals without undueadverse side effects (such as toxicity, irritation, and allergicresponse) commensurate with a reasonable benefit/risk ratio.

The terms “determining”, “measuring”, “evaluating”, “detecting”,“assessing” and “assaying” are used interchangeably herein to refer toany form of measurement, and include determining if an element ispresent or not. These terms include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present, aswell as determining whether it is present or absent.

As used herein, the term “agent” is meant to encompass any molecule,chemical entity, composition, drug, therapeutic agent, chemotherapeuticagent, or biological agent capable of preventing, ameliorating, ortreating a disease or other medical condition. The term includes smallmolecule compounds, antisense reagents, siRNA reagents, antibodies,enzymes, peptides organic or inorganic molecules, natural or syntheticcompounds and the like. An agent can be assayed in accordance with themethods of the invention at any stage during clinical trials, duringpre-trial testing, or following FDA-approval.

By the term “modulate,” it is meant that any of the mentionedactivities, are, e.g., increased, enhanced, increased, agonized (acts asan agonist), promoted, decreased, reduced, suppressed blocked, orantagonized (acts as an agonist). Modulation can increase activity morethan 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., overbaseline values. Modulation can also decrease its activity belowbaseline values. Modulation can also normalize an activity to a baselinevalue.

As used herein, the term “kit” refers to any delivery system fordelivering materials. Inclusive of the term “kits” are kits for bothresearch and clinical applications. In the context of reaction assays,such delivery systems include systems that allow for the storage,transport, or delivery of reaction reagents (e.g., oligonucleotides,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to delivery systems comprising two or more separate containersthat each contains a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides orliposomes. The term “fragmented kit” is intended to encompass kitscontaining Analyte specific reagents (ASR's) regulated under section520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limitedthereto. Indeed, any delivery system comprising two or more separatecontainers that each contains a subportion of the total kit componentsare included in the term “fragmented kit.” In contrast, a “combined kit”refers to a delivery system containing all of the components of areaction assay in a single container (e.g., in a single box housing eachof the desired components). The term “kit” includes both fragmented andcombined kits.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, and physiology.

With respect to tissue culture and embryonic stem cells, the reader maywish to refer to Teratocarcinomas and embryonic stem cells: A practicalapproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al. eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Supercharged Proteins

Supercharged proteins can be produced by changing non-conserved aminoacids on the surface of a protein to more polar or charged amino acidresidues. The amino acid residues to be modified may be hydrophobic,hydrophilic, charged, or a combination thereof. Supercharged proteinscan also be produced by the attachment of charged moieties to theprotein in order to supercharge the protein. Supercharged proteinsfrequently are resistant to aggregation, have an increased ability torefold, resist improper folding, have improved solubility, and aregenerally more stable under a wide range of conditions, includingdenaturing conditions such as heat or the presence of a detergent.

Any protein may be modified using the inventive system to produce asupercharged protein. Natural as well as unnatural proteins (e.g.,engineered proteins) may be modified. Example of proteins that may bemodified include receptors, membrane bound proteins, transmembraneproteins, enzymes, transcription factors, extracellular proteins,therapeutic proteins, cytokines, messenger proteins, DNA-bindingproteins, RNA-binding proteins, proteins involved in signaltransduction, structural proteins, cytoplasmic proteins, nuclearproteins, hydrophobic proteins, hydrophilic proteins, etc. A protein tobe modified may be derived from any species of plant, animal, and/ormicroorganism. In certain embodiments, the protein is a mammalianprotein. In certain embodiments, the protein is a human protein. Incertain embodiments, the protein is derived from an organism typicallyused in research. For example, the protein to be modified may be from aprimate (e.g., ape, monkey), rodent (e.g., rabbit, hamster, gerbil),pig, dog, cat, fish (e.g., Danio rerio), nematode (e.g., C. elegans),yeast (e.g., Saccharomyces cerevisiae), or bacteria (e.g., E. coli). Incertain embodiments, the protein is non-immunogenic. In certainembodiments, the protein is non-antigenic. In certain embodiments, theprotein does not have inherent biological activity or has been modifiedto have no biological activity. In certain embodiments, the protein ischosen based on its targeting ability. In certain embodiments, theprotein is green fluorescent protein.

Compositions and Treatments

Disclosed herein are compositions for gene editing of cells of the innerear, and any cell, damaged or otherwise, associated with hearing andhearing loss, such as, for example, stem cells, hair cells, supportingcells, neurites, strial vascularis and the like. In particular, thetargeted cells are the hair cells and supporting cells. Thecompositions, embodied herein, edit mutations associated with deafnessor disorders thereof, in hair cells and supporting cells. Hair cells areinner ear sensory cells, including inner hair cells, outer hair cells,and vestibular hair cells. Supporting cells include Deiters cells,Hensen cells, Pillar cells, inner phalangeal cells, inner border cells,Claudius cells, border cells, basal cells, interdental cells, innersulcus, spiral limbus.

Hearing loss affects a large portion of population yet no treatment isavailable beyond hearing aids and cochlear implant, both of whichprovide limited benefits. In the US alone, over 30 millions of peoplesuffer from hearing loss. The major cause of hearing loss in human isdue to irreversible loss of the inner ear sensory cells, hair cells,which are responsible for detecting sounds and sensing balance. Furtherhearing loss can be caused by defective cell types in the inner ear suchas strial vascularis and supporting cells, whose regeneration could leadto restoration of hearing.

Accordingly, embodiments are directed to protein-mediated deliverysystems to deliver the biological proteins directly to inner ear celltypes with functional consequences. With these methods, the proteinsdelivered have specific functions, and the effect is transient. Furtherdelivery of native protein lessens any potential immune response. Suchan approach would not only be valuable for inner ear delivery, but hasfar reaching effects on the delivery of proteins targeting differentdiseases, achieving specific effects in multiple organs and cell types.

For efficient protein delivery, proteins need to enter cells withsufficient amount, and are released from endosomes within cells andreach the targets. Improvement in the endosome release could havedrastic effects on the amount of protein to reach the target withenhanced biological effect. Antimicrobial or membrane destabilizingpeptides, for example, Aurein, in combination with s-GFP, can serve asan effective carrier to deliver functional proteins into mouse inner earcell types with high efficiency.

High-efficiency delivery of functional proteins in the inner ear hastremendous advantage over conventional systems, including (1). It allowsthe delivery of unlimited combinations of proteins to mammalian innerear without limitations on the size or localization of proteinsdelivered; (2). The effect will be specific; (3). The effect will betransient as delivered proteins will be degraded over time, which allowsthe modification of proteins (e.g., to reduce the response todegradation pathway) and controls the time length of function. Thedelivery system can be used to study protein functions, therapeutics forhearing recovery, hearing protection, and gene editing to correctgenetic deafness by genome-editing proteins.

In an embodiment, a method of treating deafness or disorders thereof,associated with a genetic mutation in a patient in need thereof,comprises administering to an inner ear of the patient a therapeuticallyeffective amount of a chimeric molecule comprising at least one membranedestabilizing domain and a protein or peptide fused, complexed or linkedto one or more one or more gene editing agents, transcriptionalmodulators, translational modulators, post-translational modulators,and/or modulators that regulate protein expression, function, activityor combinations thereof. The chimeric molecule targets one or moregenetic loci associated with deafness in a patient and modulatesreplication, expression, function or activity of the genetic locus inhair cells and supporting cells of the inner ear. The chimeric moleculecomprises a supercharged protein or variants thereof, for example, thesupercharged protein is green fluorescent protein (s-GFP), or variantsthereof. The gene editing agents comprise: transcriptional activators,transcriptional repressors, recombinases, nucleases, DNA-bindingproteins or nucleic acids, or combinations thereof. In some embodiments,the gene editing agents comprise: Cre recombinases, CRISPR/Casmolecules, TALE transcriptional activators, Cas9 nucleases, nickases,transcriptional regulators or combinations thereof.

In embodiments, the chimeric molecules target different chromosomalabnormalities, such as, for example, deletion, insertion, duplication,inversion and the like. The chimeric molecules are suitable therapeuticswhere bi-genic or multi-genic mutations contribute to hearing loss.CRISPR technology is well suited to target multiple genes.

In an embodiment, a membrane destabilizing domain comprises one or moreof: antimicrobial or membrane destabilizing proteins or peptides,polynucleotides, oligonucleotides, bacterial or viral (e.g. reovirusouter capsid protein or peptide, μ1; papilloma virus capsid protein orpeptide L2; etc.), antibacterial molecules, microtubules, lipids,synthetic or natural molecules. Membrane destabilization peptides areknown in the art. See, for example, Fernandez, D. I. et al., Biochim.Biophys. Acta, 2009 August; 1788(8):1630-8. Antimicrobial peptides(AMPs) are a class of membrane-active peptides that penetrate microbialmembranes to provide defense against bacteria, fungi, and viruses, oftenwith high selectivity (Zasloff, M. Nature 2002, 415, 389).

In an embodiment, the supercharged protein further comprises a membranedestabilization protein, such, as for example, aurein, which is linkedto the s-GFP or variants thereof. Accordingly, in one embodiment thechimeric molecule comprises a supercharged molecule connected to amembrane destabilization domain which in turn is connected to one ormore gene editing agents, transcriptional activators, transcriptionalrepressors, recombinases, nucleases, DNA-binding proteins or nucleicacids, or combinations thereof. In another embodiment the chimericmolecule comprises a supercharged molecule connected to a membranedestabilization domain which in turn is connected to a gene editingagent, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In another embodiment the chimeric moleculecomprises one or more membrane destabilization domains connected to oneor more gene editing agents, transcriptional activators, transcriptionalrepressors, recombinases, nucleases, DNA-binding proteins or nucleicacids, or combinations thereof. In another embodiment, the chimericmolecule comprises a membrane destabilization domain connected to asupercharged molecule which in turn is connected to one or more geneediting agents, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. One of ordinary skill in the art would understandthat there are a multitude of configurations which can be obtained.Accordingly if the letter “S” represents the supercharged domain, theletter “M” represents the antimicrobial or membrane destabilizingdomain, and the letter “X” represents the therapeutic moleculesresponsible for correcting, silencing or modifying a genetic mutation,then the molecular structure of the molecule can be S-M-X, or M-X-X, orS-M-M-X, or S-S-M-M-X, etc. In those cases where there are two or moremolecules that comprise a domain, these domains can comprise differentmolecules or they can be the same molecule. One of ordinary skill in theart would understand the possible configurations that can be possible.

In another embodiment, a chimeric molecule comprises any one or moresequences SEQ ID NOS: 1 to 95 linked to an antimicrobial or membranedestabilization domain. The antimicrobial or membrane destabilizationdomain can be connected to the supercharged domain on one end and totherapeutic molecules on the other end (e.g. upstream or downstream).

In some embodiments the chimeric molecule is encapsulated in a cationiclipid formulation.

In another embodiment, a chimeric molecule comprises any two or moreproteins, peptides or variants thereof, said proteins, peptides orvariants thereof comprising: a supercharged protein or peptide,antimicrobial or membrane destabilizing peptide, gene editing agents,transcriptional activators, transcriptional repressors, recombinases,nucleases, DNA-binding proteins or nucleic acids, or combinationsthereof.

In another embodiment, a chimeric molecule comprises any two or moreproteins, peptides or variants thereof, said proteins, peptides orvariants thereof comprising: a supercharged protein or peptide and anantimicrobial or membrane destabilizing peptide and one or more geneediting agents, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof.

In another embodiment, a chimeric molecule comprises any two or moreproteins, peptides or variants thereof, said proteins, peptides orvariants thereof comprising: an antimicrobial or membrane destabilizingdomain and one or more supercharged proteins or peptides, gene editingagents, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof.

In another embodiment, a chimeric nucleic acid molecule encoding any twoor more proteins, peptides or variants thereof, said proteins, peptidesor variants thereof comprising: a supercharged protein or peptide and anantimicrobial or membrane destabilizing peptide, and one or more geneediting agents, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof.

In some embodiments, the gene editing agents comprise: Cre recombinases,CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases,nickases, transcriptional regulators or combinations thereof.

In another embodiment, a chimeric molecule comprises a superchargedprotein or peptide and an antimicrobial or membrane destabilizingpeptide and one or more gene editing agents, transcriptional activators,transcriptional repressors, recombinases, nucleases, DNA-bindingproteins or nucleic acids, or combinations thereof. In embodiments, thegene editing agents comprise: Cre recombinases, CRISPR/Cas molecules,TALE transcriptional activators, Cas9 nucleases, nickases,transcriptional regulators or combinations thereof.

In another embodiment, a chimeric nucleic acid molecule encoding any twoor more proteins, peptides or variants thereof, said proteins, peptidesor variants thereof comprises a supercharged protein or peptide,antimicrobial or membrane destabilizing domain, gene editing agents,transcriptional activators, transcriptional repressors, recombinases,nucleases, DNA-binding proteins or nucleic acids, or combinationsthereof. In embodiments, the gene editing agents comprise: Crerecombinases, CRISPR/Cas molecules, TALE transcriptional activators,Cas9 nucleases, nickases, transcriptional regulators or combinationsthereof.

In another embodiment, a chimeric molecule comprises a nucleic acidmolecule encoding a supercharged protein or peptide and an antimicrobialor membrane destabilizing domain and one or more gene editing agents,transcriptional activators, transcriptional repressors, recombinases,nucleases, DNA-binding proteins or nucleic acids, or combinationsthereof. In embodiments, the gene editing agents comprise: Crerecombinases, CRISPR/Cas molecules, TALE transcriptional activators,Cas9 nucleases, nickases, transcriptional regulators or combinationsthereof.

In one embodiment, the nucleic acids are gRNA's. See, for example, FIG.2, SEQ ID NOS: 21-33 and SEQ ID NOS: 90-95. In one embodiment, the gRNAis SEQ ID NO: 26 (FIG. 2; PMCA2-4mut). In another embodiment, the gRNAcomprises SEQ ID NOS: 90-95.

Other examples of chimeric molecules comprising gRNA are

TMC1-1 WT (SEQ ID NO: 90):GGGACAGAACATCCCCAGGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTTMC1-2 WT (SEQ ID NO: 91):GGTGGGACAGAACATCCCCAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTTTMC1-3 WT (SEQ ID NO: 92):GGGTGGGACAGAACATCCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTTMC1-1 MUT (SEQ ID NO: 93):GGGACAGAACTTCCCCAGGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTTMC1-2 MUT (SEQ ID NO: 94):GGTGGGACAGAACTTCCCCAGGGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTTTMC1-3 MUT (SEQ ID NO: 95):GGGTGGGACAGAACTTCCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT

In another embodiment, the chimeric molecule comprises nucleic acidsequences comprising SEQ ID NOS: 21-33, 90-95 or combinations thereof.In another embodiment, the chimeric molecule comprises SEQ ID NOS: 26,94, 95 or combinations thereof.

In another embodiment, a method of correcting, silencing or modifying agenetic mutation associated with deafness or disorders thereof, in apatient, comprises administering to a patient's inner ear, a cationicliposome encapsulating a therapeutically effective amount of a moleculecomprising a supercharged protein or peptide fused, complexed or linkedto one or more one or more gene editing agents, transcriptionalmodulators, translational modulators, post-translational modulators,and/or modulators that regulate protein expression, function, activityor combinations thereof. The molecule targets one or more genetic locihaving a mutation associated with deafness or disorders thereof, in apatient, wherein the molecule corrects, silences or modifies a geneticmutation in hair cells and supporting cells of the inner ear.

In another embodiment, a method of correcting, silencing or modifying agenetic mutation associated with deafness or disorders thereof, in apatient, comprises administering to a patient's inner ear, a cationicliposome encapsulating a therapeutically effective amount of a moleculecomprising a supercharged protein or peptide and an antimicrobial ormembrane destabilizing domain fused, complexed or linked to one or moreone or more gene editing agents, transcriptional modulators,translational modulators, post-translational modulators, and/ormodulators that regulate protein expression, function, activity orcombinations thereof. The molecule targets one or more genetic locihaving a mutation associated with deafness or disorders thereof, in apatient, wherein the molecule corrects, silences or modifies a geneticmutation in hair cells and supporting cells of the inner ear.

In another embodiment, a method of correcting, silencing or modifying agenetic mutation in hair cells and/or supporting cells in vitro or invivo, comprising: contacting a hair cell or supporting cell oradministering to a patient's inner ear, a therapeutically effectiveamount of a molecule comprising an antimicrobial or membranedestabilizing domain, a supercharged protein or peptide fused, complexedor linked to one or more one or more gene editing agents,transcriptional modulators, translational modulators, post-translationalmodulators, and/or modulators that regulate protein expression,function, activity or combinations thereof. The molecule targets one ormore genetic loci having a mutation associated with deafness ordisorders thereof, in a patient, wherein the molecule corrects, silencesor modifies a genetic mutation in hair cells and supporting cells of theinner ear. For gene editing such as CRISPR, Cas9 (protein) is complexedwith gRNA (guide RNA, nucleic acid) with/without a template by liposome.Such complex can then be delivered to inner ear for gene editing. Forthe disruption of mutation, only gRNA without template is needed;whereas for mutation repair, both a gRNA and a template are needed; inaddition to Cas9.

In another embodiment, a method of delivering a therapeutic molecule tocells of an inner ear of a patient, comprises administering to the innerear of a patient a chimeric molecule comprising at least one protein orpeptide fused, complexed or linked to one or more anionic molecules. Inan embodiment, the cells of the inner ear comprise hair cells,supporting cells, or combinations thereof. In an embodiment, thechimeric molecule comprises one or more gene editing agents,transcriptional modulators, translational modulators, post-translationalmodulators, and/or modulators that regulate protein expression,function, activity or combinations thereof. The gene editing agentscomprise: transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In an embodiment, the anionic molecules comprise:oligonucleotides, polynucleotides, proteins, peptides, peptide nucleicacids (PNA), synthetic molecules or combinations thereof. Theoligonucleotides or polynucleotides comprise: ribonucleic acids (RNA),deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modifiedRNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA),interference RNA, mRNA, nucleic acid sequences comprising one or moremodified nucleobases or backbones, or combinations thereof. In otherembodiments, the chimeric molecule comprises a supercharged protein orvariants thereof. An example of a supercharged protein is a fluorescentprotein, or variants thereof. The chimeric molecule further comprises amembrane destabilizing protein, for example, aurein.

In some embodiments, a composition comprises a cationic lipidencapsulating one or more chimeric molecules. These chimeric moleculescomprise one or more proteins or peptides fused, complexed or linked toone or more anionic molecules. The anionic molecules can vary as long asthey comprise one or more anionic domains or bind to an anionic nucleicacid domain. It is preferred that the anionic molecules confer anoverall net negative charge to the chimeric molecule. Without wishing tobe bound by theory, it was hypothesized that proteins that areengineered to be highly negatively charged or that are naturally highlyanionic may be able to take advantage of the same electrostatics-drivencomplexation and encapsulation used by cationic liposomal reagents fornucleic acid delivery. While few proteins natively possess the densityof negative charges found in the phosphate backbone of nucleic acids, itwas speculated that translational fusion to, or non-covalentcomplexation with, a polyanionic molecule may render the resultingprotein or protein complex sufficiently anionic to be efficientlycomplexed by common cationic lipid reagents. The results for the work,described in the Examples section which follows, showed that deliveryefficiency depends on the net charge of the fusion protein, and nativelyanionic peptide tags such as 3×FLAG and VP64 can also enablelipid-mediated protein delivery.

Accordingly, in some embodiments, the anionic molecules comprise:oligonucleotides, polynucleotides, proteins, peptides, peptide nucleicacids (PNA), synthetic molecules or combinations thereof. In someembodiments, the oligonucleotides or polynucleotides comprise:ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA orDNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA),short guide RNA (sgRNA), a short interfering RNA (siRNA), a micro,interfering RNA (miRNA), a small, temporal RNA (stRNA), a short, hairpinRNA (shRNA), mRNA, nucleic acid sequences comprising one or moremodified nucleobases or backbones, or combinations thereof.

In embodiments, the one or more proteins or peptides of the chimeric oranionic molecule can possess any charge as long as the overall netcharge of the chimeric molecule is anionic. Accordingly, in embodiments,the proteins or peptides are cationic, anionic or are neutrally charged.Examples of proteins or peptides of the chimeric molecule which can becomplexed or linked to the polyanionic molecule or domain comprise:enzymes, hormones, chemotherapeutic agents, immunotherapeutic agents,gene editing agents, synthetic molecules or combinations thereof.

In some embodiments, the protein or peptide is a therapeutic agent fordelivery to a specific target. The target can be any desiredintracellular target. In some embodiments, the target is a nucleic acidsequence or gene. In embodiments where it is desired to manipulate,modulate or edit a gene, the protein or peptide is a gene or genomeediting agent. In some embodiments, the gene editing agents comprise:transcriptional activators, transcriptional repressors, transcriptionfactors, enhancer modulating molecules, recombinases, nucleases, nucleicacid binding-proteins, nucleic acid binding-polynucleotides oroligonucleotides, DNA-binding proteins or DNA-binding nucleic acids, orcombinations thereof. In some embodiments, the target is a protein orpeptide. Accordingly, in some embodiments, the chimeric or anionicmolecule comprises one or more gene editing agents, transcriptionalmodulators, translational modulators, post-translational modulators,and/or modulators that regulate protein expression, function, activityor combinations thereof.

In other embodiments, the chimeric molecule further comprises one ormore detectable labels, anions, radiolabels, tags, targeting agents,negatively charged proteins or peptides, or combinations thereof. Thesemolecules can be selected based on the user's desired goal, e.g. fordiagnostic or research purposes, or to increase the anionic charge,targeting signals and the like. Accordingly, a liposomal formulation forcomplexing protein and nucleic acid (e.g. transcription factors withtheir target binding region as oligonucleotides) for inner ear celltypes delivery in vivo, is used to treat deafness or associateddisorders thereof as the chimeric molecule can be tailored forregeneration (e.g. hair cell and auditory neuron regeneration), repair(e.g. re-establishment of connections between hair cells and neurons forhearing recovery) and prevention.

In other embodiments, a chimeric molecule comprises at least one proteinor peptide fused, complexed or linked to one or more anionic molecules.Preferably, the one or more anionic molecules comprise one or moreanionic domains or bind to an anionic nucleic acid domain. Inembodiments, the chimeric molecule comprises an overall net negativecharge. In some embodiments, the anionic molecules comprise:oligonucleotides, polynucleotides, proteins, peptides, peptide nucleicacids (PNA), synthetic molecules or combinations thereof. In someembodiments, the oligonucleotides or polynucleotides comprise:ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA orDNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA),short guide RNA (sgRNA), a short interfering RNA (siRNA), a micro,interfering RNA (miRNA), a small, temporal RNA (stRNA), a short, hairpinRNA (shRNA), mRNA, nucleic acid sequences comprising one or moremodified nucleobases or backbones, or combinations thereof. The chimericmolecule also comprises one or more proteins or peptides which arecationic, anionic or are neutrally charged. Examples of proteins includewithout limitation: enzymes, hormones, chemotherapeutic agents,immunotherapeutic agents, genome or gene editing agents, syntheticmolecules or combinations thereof. The gene or genome editing agentscomprise: transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In other embodiments, the chimeric moleculeoptionally comprises one or more detectable labels, radiolabels, tags,anions, targeting agents or combinations thereof.

In other embodiments, a cationic liposome encapsulates an anionicmolecule comprising a protein or peptide complexed, fused or linked to anegatively charged molecule. In some embodiments, the negatively chargedmolecule comprises oligonucleotides, polynucleotides, proteins,peptides, peptide nucleic acids (PNA), synthetic molecules orcombinations thereof. In other embodiments, the polynucleotide oroligonucleotide is a guide RNA. In some embodiments, the protein orpeptide is a negatively charged protein. In yet other embodiments, theone or more proteins or peptides are cationic, anionic or are neutrallycharged. In yet another embodiment, the negatively charged protein isfused or linked to one or more proteins or peptides. In someembodiments, the protein or peptide comprises: enzymes, hormones,chemotherapeutic agents, immunotherapeutic agents, gene editing agents,synthetic molecules or combinations thereof. In some embodiments, thegene editing agents comprise: transcriptional activators,transcriptional repressors, transcription factors, enhancer modulatingmolecules, recombinases, nucleases, nucleic acid binding-proteins,nucleic acid binding-polynucleotides or oligonucleotides, DNA-bindingproteins or DNA-binding nucleic acids, or combinations thereof. Examplesof these gene editing agents comprise: Cre recombinases, CRISPR/Casmolecules, TALE transcriptional activators, Cas9 nucleases, nickases,transcriptional regulators or combinations thereof. The anionic moleculeoptionally comprises one or more detectable labels, radiolabels, tags,negatively charged proteins or peptides, anions, targeting agents orcombinations thereof.

In some embodiments, a molecule comprises any one or more sequencescomprising SEQ ID NOS: 1 to 95. In another embodiment, a moleculecomprises any one or more sequences comprising SEQ ID NOS: 1 to 95,wherein each sequence further comprises one or more antimicrobial ormembrane destabilizing domains, for example, aurein or fragmentsthereof.

In other embodiments, the liposome comprises one or more cationiclipids, modified lipids or combinations thereof.

In some embodiments, a liposome encapsulating one or more moleculesembodied herein comprises a liposome, a nanoliposome, a niosome, amicrosphere, a nanosphere, a nanoparticle, a micelle, or an archaeosome.

Modified Proteins or Peptides:

Hybrid proteins comprising a polypeptide or fragment thereof may belinked to other types of polypeptides, for example, a negativelysupercharged protein, e.g. green fluorescent protein in addition to areporter polypeptide, or in lieu of a reporter polypeptide. Theseadditional polypeptides may be any amino acid sequence useful for thepurification, identification, overall charge of the protein or peptide,and/or therapeutic or prophylactic application of the peptide. Inaddition, the additional polypeptide can be a signal peptide, ortargeting peptide, etc.

In some cases, the other additions, substitutions or deletions mayincrease the stability (including but not limited to, resistance toproteolytic degradation) of the polypeptide or increase affinity of thepolypeptide for its appropriate receptor, ligand and/or bindingproteins. In some cases, the other additions, substitutions or deletionsmay increase the solubility of the polypeptide. In some embodimentssites are selected for substitution with a naturally encoded ornon-natural amino acid in addition to another site for incorporation ofa non-natural amino acid for the purpose of increasing the polypeptidesolubility following expression in recombinant host cells. In someembodiments, the polypeptides comprise another addition, substitution,or deletion that modulates affinity for the associated ligand, bindingproteins, and/or receptor, modulates (including but not limited to,increases or decreases) receptor dimerization, stabilizes receptordimers, modulates circulating half-life, modulates release orbio-availability, facilitates purification, or improves or alters aparticular route of administration. Similarly, the non-natural aminoacid polypeptide can comprise chemical or enzyme cleavage sequences,protease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including but not limited to, biotin) that improvedetection (including but not limited to, GFP), purification, transportthrough tissues or cell membranes, prodrug release or activation, sizereduction, or other traits of the polypeptide.

The methods and compositions described herein include incorporation ofone or more non-natural amino acids into a polypeptide. One or morenon-natural amino acids may be incorporated at one or more particularpositions which does not disrupt activity of the polypeptide. This canbe achieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with non-natural ornatural hydrophobic amino acids, bulky amino acids with non-natural ornatural bulky amino acids, hydrophilic amino acids with non-natural ornatural hydrophilic amino acids) and/or inserting the non-natural aminoacid in a location that is not required for activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-natural amino acidwithin the polypeptide. Any position of the polypeptide chain issuitable for selection to incorporate a non-natural amino acid, andselection may be based on rational design or by random selection for anyor no particular desired purpose. Selection of desired sites may bebased on producing a non-natural amino acid polypeptide (which may befurther modified or remain unmodified) having any desired property oractivity, including but not limited to agonists, super-agonists, partialagonists, inverse agonists, antagonists, receptor binding modulators,receptor activity modulators, modulators of binding to binder partners,binding partner activity modulators, binding partner conformationmodulators, dimer or multimer formation, no change to activity orproperty compared to the native molecule, or manipulating any physicalor chemical property of the polypeptide such as solubility, aggregation,or stability. For example, locations in the polypeptide required forbiological activity of a polypeptide can be identified using methodsincluding, but not limited to, point mutation analysis, alanine scanningor homolog scanning methods. Residues other than those identified ascritical to biological activity by methods including, but not limitedto, alanine or homolog scanning mutagenesis may be good candidates forsubstitution with a non-natural amino acid depending on the desiredactivity sought for the polypeptide. Alternatively, the sites identifiedas critical to biological activity may also be good candidates forsubstitution with a non-natural amino acid, again depending on thedesired activity sought for the polypeptide. Another alternative wouldbe to make serial substitutions in each position on the polypeptidechain with a non-natural amino acid and observe the effect on theactivities of the polypeptide. Any means, technique, or method forselecting a position for substitution with a non-natural amino acid intoany polypeptide is suitable for use in the methods, techniques andcompositions described herein.

Modified Oligonucleotides:

Examples of some oligonucleotides envisioned for this invention includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. In some embodiments, modified oligonucleotidescomprise those with phosphorothioate backbones and those with heteroatombackbones, CH₂—NH—O—CH₂, CH, —N(CH₃)—O—CH₂ [known as amethylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH). The amide backbonesdisclosed by De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374) arealso embodied herein. In some embodiments, the oligonucleotides havingmorpholino backbone structures (Summerton and Weller, U.S. Pat. No.5,034,506), peptide nucleic acid (PNA) backbone wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleobases being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone (Nielsen et al.Science 1991, 254, 1497). Oligonucleotides may also comprise one or moresubstituted sugar moieties. Oligonucleotides may also have sugarmimetics such as cyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N₆ (6-aminohexyl)adenine and2,6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman & Co.,San Francisco, 1980, pp 75-77; Gebeyehu, G., et al. Nucl. Acids Res.1987, 15:4513). A “universal” base known in the art, e.g., inosine maybe included. 5-Me-C substitutions have been shown to increase nucleicacid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T.and Lebleu, B., eds., Antisense Research and Applications, CRC Press,Boca Raton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid(Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al. Ann. N.Y. Acad. Sci. 1992,660, 306; Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327;Svinarchuk et al. Biochimie 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.Tetrahedron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18,3777), a polyamine or a polyethylene glycol chain (Manoharan et al.Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid(Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). Oligonucleotidescomprising lipophilic moieties and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

Labeled Molecules:

In another preferred embodiment, the chimeric molecules can be labeled.Uses include therapeutic and imaging for diagnostic and prognosticpurposes. The label may be a radioactive atom, an enzyme, or achromophore moiety. Methods for labeling antibodies have been described,for example, by Hunter and Greenwood, Nature, 144:945 (1962) and byDavid et al. Biochemistry 13:1014-1021 (1974). Additional methods forlabeling antibodies have been described in U.S. Pat. Nos. 3,940,475 and3,645,090. Methods for labeling oligonucleotide probes have beendescribed, for example, by Leary et al. Proc. Natl. Acad. Sci. USA(1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435;Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al.Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem.(1984) 138:267.

The label may be radioactive. Some examples of useful radioactive labelsinclude ³²P ¹²⁵I, ¹³¹I and ³H. Use of radioactive labels has beendescribed in U.K. 2,034,323, U.S. Pat. Nos. 4,358,535, and 4,302,204.

Some examples of non-radioactive labels include enzymes, chromophores,atoms and molecules detectable by electron microscopy, and metal ionsdetectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectablechange in a substrate. Some useful enzymes and their substrates include,for example, horseradish peroxidase (pyrogallol and o-phenylenediamine),β-galactosidase (fluorescein β-D-galactopyranoside), and alkalinephosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium). The use of enzymatic labels has been described in U.K.2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci. USA, 47,1981-1991 (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent,and bioluminescent molecules, as well as dyes. Some specificchromophores useful in the present invention include, for example,fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone,luminol.

The labels may be conjugated to the chimeric molecule by methods thatare well known in the art. The labels may be directly attached through afunctional group on the probe. The probe either contains or can becaused to contain such a functional group. Some examples of suitablefunctional groups include, for example, amino, carboxyl, sulfhydryl,maleimide, isocyanate, isothiocyanate. Alternatively, labels such asenzymes and chromophores may be conjugated to the antibodies ornucleotides by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like.

In another preferred embodiment, the chimeric fusion molecules of theinvention can be used for imaging. In imaging uses, the complexes arelabeled so that they can be detected outside the body. Typical labelsare radioisotopes, usually ones with short half-lives. The usual imagingradioisotopes, such as ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)TC, ¹⁸⁶Re, ⁶⁴Cu,⁶⁷Cu, ²¹²Bi, ²¹³Bi, ⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹⁸F, ³H, ¹⁴C, ³⁵S or ³²P can beused. Nuclear magnetic resonance (NMR) imaging enhancers, such asgadolinium-153, can also be used to label the complex for detection byNMR. Methods and reagents for performing the labeling, either in thepolynucleotide or in the protein moiety, are considered known in theart.

Reporter genes useful in the present invention include acetohydroxy acidsynthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ),beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), red fluorescent protein (RFP), yellowfluorescent protein (YFP), cyan fluorescent protein (CFP), horseradishperoxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopinesynthase (OCS), and derivatives thereof. Multiple selectable markers areavailable that confer resistance to ampicillin, bleomycin,chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin,methotrexate, phosphinothricin, puromycin, and tetracycline. Methods todetermine modulation of a reporter gene are well known in the art, andinclude, but are not limited to, fluorometric methods (e.g. fluorescencespectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescencemicroscopy), antibiotic resistance determination.

Methods of Treatment

The compositions and molecules embodied herein are useful in thosediseases and conditions that would benefit from protein therapeutics. Insome embodiments, a method of treatment comprises administering to apatient an effective amount of cationic liposome encapsulating achimeric molecule embodied herein. In other embodiments, the moleculecomprises one or more sequences set forth as SEQ ID NOS: 1 to 32.

In another embodiment, a method of treatment comprises administering toa patient a therapeutically effective amount of a chimeric moleculecomprising any two or more proteins, peptides or variants thereof, saidproteins, peptides or variants thereof comprising: a superchargedprotein or peptide, antimicrobial or membrane destabilizing peptide,transcriptional activators, transcriptional repressors, recombinases,nucleases, DNA-binding proteins or nucleic acids, or combinationsthereof. In embodiments, the gene editing agents comprise: Crerecombinases, CRISPR/Cas molecules, TALE transcriptional activators,Cas9 nucleases, nickases, transcriptional regulators or combinationsthereof.

In another embodiment, a method of treatment comprises administering toa patient a therapeutically effective amount of a chimeric moleculecomprising any two or more proteins, peptides or variants thereof, saidproteins, peptides or variants thereof comprising: a superchargedprotein or peptide and an antimicrobial or membrane destabilizingpeptide and one or more transcriptional activators, transcriptionalrepressors, recombinases, nucleases, DNA-binding proteins or nucleicacids, or combinations thereof. In embodiments, the gene editing agentscomprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptionalactivators, Cas9 nucleases, nickases, transcriptional regulators orcombinations thereof.

In another embodiment, a method of treatment comprises administering toa patient a therapeutically effective amount of a chimeric moleculecomprising any two or more proteins, peptides or variants thereof, saidproteins, peptides or variants thereof comprising: an antimicrobial ormembrane destabilizing domain and one or more supercharged proteins orpeptides, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In embodiments, the gene editing agents comprise:Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators,Cas9 nucleases, nickases, transcriptional regulators or combinationsthereof.

In another embodiment, a method of treatment comprises administering toa patient a therapeutically effective amount of a chimeric moleculecomprising an antimicrobial or membrane destabilizing domain, one ormore supercharged proteins or peptides, transcriptional activators,transcriptional repressors, recombinases, nucleases, DNA-bindingproteins or nucleic acids, or combinations thereof. In embodiments, thegene editing agents comprise: Cre recombinases, CRISPR/Cas molecules,TALE transcriptional activators, Cas9 nucleases, nickases,transcriptional regulators or combinations thereof.

In another embodiment, a method of treatment comprises administering toa patient a therapeutically effective amount of a chimeric nucleic acidmolecule encoding any two or more proteins, peptides or variantsthereof, said proteins, peptides or variants thereof comprising: asupercharged protein or peptide, antimicrobial or membrane destabilizingdomain, transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof. In embodiments, the gene editing agents comprise:Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators,Cas9 nucleases, nickases, transcriptional regulators or combinationsthereof.

In other embodiments, the method of treating hearing loss or deafnessusing the liposomal formulation for complexing protein and nucleic acidembodied herein (e.g. transcription factors with their target bindingregion as oligonucleotides) for inner ear cell type delivery in vivo,comprises regeneration (e.g. hair cell and auditory neuronregeneration), repair (e.g. re-establishment of connections between haircells and neurons for hearing recovery) and prevention.

Hearing Loss or Deafness and Associated Disorders: One in 1000 newbornssuffers from genetic deafness. Over 80 deafness genes have beenidentified, and additional 200-300 deafness genes remain to bediscovered. Despite the tremendous progress, there is no treatment forany genetic deafness. Thus there are urgent needs to develop treatmentthat targets different types of genetic deafness.

There two main categories of genetic deafness: recessive deafness thatis generally congenital; and dominant deafness that is mainlyprogressive. For recessive deafness, delivery and continuous expressionof a normal copy of mutant gene could compensate for lost function forhearing recovery. Adeno-associated virus (AAV) based gene therapy hasbeen the choice to be developed as treatment for recessive deafness, dueto its long-term expression pattern and good safety record. AAV vectorshowever can only accommodate inserts less than 4.5 kb, whereas manydeafness genes are much larger in size, thus severely limitingusefulness of AAV. For dominant deafness gene delivery will unlikelywork.

Non-inherited abnormalities of the inner ear, such as the Mondinimalformation, account for roughly 20% of congenital sensorineuraldeafness. The bulk of the remaining (genetic) deafness is non-syndromic,meaning that it does not have any obvious distinguishing features.

Most non-syndromic hearing losses are caused by connexin gene mutations.In the mammals, at least 20 connexin subtypes have been identified inmouse and human genomics. Connexin genes encode gap junctional channels,which connect two adjacent cells allowing passage of cytoplasmic ionsand small molecules up to 1.2 kDa. In the mammalian inner ear,connexin26 (Cx26) and Cx30 are predominant isoforms. Cx26 mutation caninduce a high incidence of hearing loss, responsible for 70 to 80percent of nonsyndromic hearing loss in children.

Non-Syndromic Deafness:

Nonsyndromic means that deafness occurs in isolation, without otherassociated disorders. About 80% of genetic hearing loss isnon-syndromic. Between 1992 and 2001, 38 loci for autosomal dominantnon-syndromic deafness have been mapped and 11 genes have beenidentified. Autosomal dominant loci are called DFNA, autosomal recessiveas DFNB, and X-linked as DFN.

Non-syndromic deafness is highly heterogeneous, but mutations in theconnexin-26 molecule (gap junction protein, gene GJB2) account for about49% of patients with non-syndromic deafness and about 37% of sporadiccases. About 1 in 31 individuals of European extraction are likelycarriers.

Autosomal Dominant (DFNA):

Autosomal dominant deafness is passed directly through generations. Itis often possible to identify an autosomal dominant pattern throughsimple inspection of the family tree. Examples of autosomal dominantdeafness are missense mutation in COL11A2 (DFNA13) and in the TMC1 gene.COL11A2 encodes a chain of type XI collagen whereas TMC1 encodes a haircell channel protein.

Autosomal Recessive (DFNB):

Autosomal recessive disorders require a gene from both the mother andfather.

Syndromic Deafness:

Syndromic deafness, which accounts for the remaining 20% of congenitaldeafness, comprises an immensely complicated interlinked set ofdisorders. The descriptions here are only to give the general flavor ofthe diseases and are not meant to include all features of the disorders.In most cases, an Online Mendelian Inheritance in Man (OMIM) databaselink to the main type of the genetic disorder is provided. This databaseis a catalog of human genes and genetic disorders.

Alport Syndrome:

Alport syndrome is caused by mutations in COL4A3, COL4A4 or COL4A5. Theclassic phenotype is renal failure and progressive sensorineuraldeafness.

Branchio-Oto-Renal Syndrome:

Branchio-oto-renal syndrome is caused by mutations in EYA1, a gene of 16exons within a genomic interval of 156 kB. This syndrome ischaracterized by hearing disturbances and cataract, branchial cleftfistulae, and preauricular pits. Mondini malformations and relateddysplasias may occur.

X-Linked Charcot Marie Tooth (CMT):

The dominantly form of X-linked CMT is caused by a mutation in theconnexin 32 gene mapped to the Xq13 locus. Usual clinical signs consistof a peripheral neuropathy combined with foot problems and “champagnebottle” calves.

As noted above, the connexin gene is also associated with a largepercentage of cases of non-syndromic deafness. There are several otherassociated neuropathies and deafness syndromes. Autosomal recessivedemyelinating neuropathy, autosomal dominant hereditary neuropathiestype I and II, and X-linked hereditary axonal neuropathies with mentalretardation are all associated with deafness.

Goldenhar's Syndrome:

Oculoauriculovertebral dysplasia (OAVD) or Goldenhar's syndrome wasoriginally described in 1881. It includes a complex of featuresincluding hemifacial microtia, otomandibular dysostosis, epibulbarlipodermoids, coloboma, and vertebral anomalies that stem fromdevelopmental vascular and genetic field aberrations. It has diverseetiologies and is not attributed to a single genetic locus. Theincidence is roughly 1 in 45,000.

Jervell and Lange-Nielsen Syndrome:

Jervell and Lange-Nielsen Syndrome is associated with cardiacarrhythmias. There is, by prolongation of the QT interval, torsade dePointe arrhythmias (turning of the points, in reference to the apparentalternating positive and negative QRS complexes), sudden syncopalepisodes, and severe to profound sensorineural hearing loss.

Mohr-Tranebjaerg Syndrome (DFN-1):

Mohr-Tranebjaerg syndrome (DFN-1) is an X-linked recessive syndromichearing loss characterized by postlingual sensorineural deafness inchildhood, followed by progressive dystonia, spasticity, dysphagia andoptic atrophy. The syndrome is caused by a mutation thought to result inmitochondrial dysfunction. It resembles a spinocerebellar degenerationcalled Fredreich's ataxia which also may exhibit sensorineural hearingloss, ataxia and optic atrophy. The cardiomyopathy characteristic ofFreidreich's ataxia is not seen in Mohr-Tranebjaerg syndrome.

Norrie Disease:

Classic features of Norrie Disease include specific ocular symptoms(pseudotumor of the retina, retinal hyperplasia, hypoplasia and necrosisof the inner layer of the retina, cataracts, phthisis bulbi),progressive sensorineural hearing loss, and mental disturbance, althoughless than one-half of patients are hearing impaired or mentallyretarded.

Pendred Syndrome:

Pendred Syndrome is deafness associated with thyroid disease (goiter).

Stickler Syndrome:

Stickler syndrome is caused by mutations in COL11. It is characterizedby hearing impairment, midface hypoplasia, progressive myopia in thefirst year of life, and arthropathy.

Treacher Collins Syndrome:

Treacher Collins syndrome (OMIM entry TCOF1) is characterized bycoloboma of the lower eyelid (the upper eyelid is involved in Goldenharsyndrome), micrognathia, microtia, hypoplasia of the zygomatic arches,macrostomia, and inferior displacement of the lateral canthi withrespect to the medial canthi.

Waardenburg Syndrome:

The clinical symptoms of Waardenburg Syndrome (WS) type I and II includelateral displacement of the inner canthus of each eye, pigmentaryabnormalities of hair, iris, and skin (often white forelock andheterochromia iridis), and sensorineural deafness. The combination of WStype I characteristics with upper limb abnormalities has been calledKlein-Waardenburg syndrome or WS type III. The combination ofrecessively inherited WS type II characteristics with Hirschsprungdisease has been called Waardenburg-Shah syndrome or WS type IV.

Usher Syndrome:

Usher syndrome is characterized by hearing impairment and retinitispigmentosa. Usher syndrome can be classified into three different typeson the basis of clinical findings. In type I, there is both hearingimpairment and vestibular impairment. In type II, there is hearingimpairment without vestibular impairment. In type III, there arevariable amounts of vestibular impairment.

Mitochondrial Disorders:

Hearing loss is common in mitochondrial disorders including MELAS(mitochondrial encephalomyopathy, lactic acidosis, and stroke likeepisodes), Kearns-Sayre syndrome and MERRF (myoclonic epilepsy withragged red fibers). These disorders are caused by mutations inmitochondrial DNA, and are characterized by muscular weakness, anabnormal muscle biopsy with “ragged red” fibers, and a variety of otherfindings that define the specific clinical phenotype. In MELAS, it wasreported that the hearing loss is caused by cochlear damage. Itresembles presbyacusis in that it is generally symmetrical, gradual, andaffects the higher frequencies first. Others have also reported hearingloss associated with mitochondrial mutations. Mitochondrial DNAmutations accumulate naturally during life and are presently implicatedas an important cause of normal aging. Mitochondrial defects have beenreported to cause both unusual sensitivity to aminoglycosides as well asnon-syndromic sensorineural deafness.

Non-Inherited Congenital Deafness:

These types of abnormalities account for roughly 20% of congenitaldeafness, the remainder being genetic in origin.

Mondini Dysplasia:

The normal cochlea has two and one-half turns. A cochlear malformationconsists of a membranous abnormality, a bony abnormality, or acombination of these two. If cochlear development is arrested in theembryo, a common cavity may occur instead of the snail like cochlea.This is called the Mondini dysplasia or malformation.

Often accompanying the Mondini dysplasia is abnormal communicationbetween the endolymphatic and perilymphatic spaces of the inner ear andsubarachnoid space. It is usually caused by a defect in the cribiformarea of the lateral end of the internal auditory canal, presumablybecause of this abnormal channel, perilymphatic fistulae are more commonin this disorder.

A related anomaly and more severe syndrome, the CHARGE association,consists of coloboma, heart disease, choanal atresia, retardeddevelopment, genital hypoplasia, ear anomalies including hypoplasia ofthe external ear and hearing loss. These individuals have a Mondini typedeformity and absence of semicircular canals.

Enlarged Vestibular Aqueduct Syndrome:

Enlarged Vestibular Aqueduct Syndrome is defined on the CT scan as adiameter greater than or equal to 1.5 mm measured midway between theoperculum and the common crus.

Recently CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats) endonuclease gene editing has been developed with potential torevolutionize genetic therapy. CRISPR uses Cas9 and guide RNA to targetany genomic sequence for specific cleavage, resulting disruption orrepair of any gene. The process applies to mutant genes regardless thenature of mutations (recessive or dominant), with permanent correctionto restore normal gene function.

Conventional approaches with CRSIP involve the use of viral vehicle todeliver Cas9 and guide RNAs (sgRNA, a template homologous to the targetgenomic region of 20-29 bp) to cells for gene editing. However the viralgenome will remain permanently inside cells (for inner ear it means thewhole life) with uncertain consequences (e.g. immunogenic response,potential recombination). In addition the efficiency of CRISPR mediatedtargeted cleavage in vivo has been relatively low (less than 5%).

A major improvement over previous methods is to directly deliver proteinand nucleic acid complex into cells for the CRISPR mediated geneediting. This approach would allow transient delivery of proteins andnucleic acids, which will be degraded after their function, thuslimiting possible adverse effect due to long-term presence of both incells. Delivery of the combination of proteins with nucleic acids hasnot been achieved in vivo or in vitro.

Nucleic acid deliveries based on cationic lipid formulations have beenused widely with high efficiency. The lipid bilayer of liposome protectsthe encapsulated nucleic acids from degradation and can preventneutralization by antibodies. Significantly, fusion of liposomes withthe endosomal membrane during endosome maturation can enable theefficient endosomal escape of cationic lipid-delivered cargo. As somenatural proteins or proteins with modifications can be highly negative(anionic), it is possible to use liposomes based vehicles to deliverproteins into cells directly with high efficiency. It is furtherpossible to combine the delivery of anionic proteins and nucleic acids(which is anionic) together with liposomes.

Accordingly, in some embodiments, a method of treating deafnessassociated with a genetic mutation in a patient in need thereof,comprises administering to the patient a therapeutically effectiveamount of a chimeric molecule comprising at least one protein or peptidefused, complexed or linked to one or more anionic molecules. Thechimeric molecule targets one or more genetic loci associated withdeafness in a patient and modulates replication, expression, function oractivity of the genetic locus. The genotypic variations that can conferabnormal phenotypes, e.g. deafness, comprise: mutations, insertions,deletions, substitutions or combinations thereof wherein the abnormalgene is expressed. In embodiments, the chimeric molecule comprises oneor more gene editing agents for repression of the genetic locusassociated with deafness in a patient. These gene editing agentscomprise: transcriptional activators, transcriptional repressors,recombinases, nucleases, DNA-binding proteins or nucleic acids, orcombinations thereof.

In embodiments, the anionic molecules comprise: oligonucleotides,polynucleotides, proteins, peptides, peptide nucleic acids (PNA),synthetic molecules or combinations thereof. Examples ofoligonucleotides or polynucleotides include: ribonucleic acids (RNA),deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modifiedRNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA),interference RNA, mRNA, nucleic acid sequences comprising one or moremodified nucleobases or backbones, or combinations thereof.

In embodiments, the chimeric molecule is encapsulated in a cationicliposome and is administered to a patient's inner ear.

In another embodiment, a method of treating a patient suffering fromdeafness due to a genetic mutation comprises: administering to apatient's inner ear, a cationic liposome encapsulating a therapeuticallyeffective amount of an anionic molecule comprising a protein or peptidecomplexed, fused or linked to a negatively charged molecule. In theseembodiments, the chimeric molecule targets one or more genetic lociassociated with deafness in a patient and modulates replication,expression, function or activity of the genetic locus. These geneticloci associated with deafness comprise: mutations, insertions,deletions, substitutions or combinations thereof. The anionic moleculecomprises one or more gene editing agents for repression of a geneticlocus associated with deafness in a patient. Examples of these geneediting agents comprise: transcriptional activators, transcriptionalrepressors, transcription factors, enhancer modulating molecules,recombinases, nucleases, nucleic acid binding-proteins, nucleic acidbinding-polynucleotides or oligonucleotides, DNA-binding proteins orDNA-binding nucleic acids, or combinations thereof.

Non-exhaustive examples of mutations in genes that cause, for example,nonsyndromic deafness, include, without limitation, mutations in theACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, ESPN, EYA4, GJB2, GJB6,GRXCR1, KCNQ4, MYO3A, MYO15A, MYO6, MYO7A, OTOF, OTOA, PCDH15, POU3F4,RDX, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, USH1C, WFS1 and WHRNgenes cause nonsyndromic deafness, with weaker evidence currentlyimplicating genes CCDC50, DIAPH1, DSPP, ESRRB, GJB3, GRHL2, GRXCR1, HGF,LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR96, MYH14, MYH9, MYO1A, MYO3A,OTOA, PJVK, POU4F3, PRPS1, PTPRQ, RDX, SERPINB6, SIX1, SLC17A8, TPRN,TRIOBP, and WHRN.

Accordingly, any one or more genes or genetic loci associated withdeafness can be targeted. In other embodiments, the molecules embodiedherein are administered to treat patients suffering from diseases ordisorders associated with deafness. Examples of these diseases ordisorders include: tinnitus, hyperscusis, ADHD.

In some embodiments, the gene editing agents comprise: Cre recombinases,CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases,nickases, transcriptional regulators or combinations thereof.

In other embodiments, the anionic molecule comprises any one or moresequences having a sequence identity of at least about 75% to sequencesset forth as SEQ ID NOS: 1 to 95. In another embodiment, the anionicmolecule comprises one or more antimicrobial or membrane destabilizingdomains.

In another embodiment, the one or more sequences are set forth as SEQ IDNOS: 1 to 95. In another embodiment, the one or more sequences compriseSEQ ID NOS: 1 to 95 linked, fused, complexed with one or moreantimicrobial or membrane destabilizing domains.

In other embodiments, the chimeric molecules or the encapsulatedchimeric or anionic molecules are administered in a pharmaceuticalcomposition.

Pharmaceutical Compositions:

The types and amounts of chimeric molecules for use as therapeuticcompounds may be believed to have therapeutic activity on the basis ofany information available to the artisan. For example, a prototypecompound may be believed to have therapeutic activity on the basis ofinformation contained in the Physician's Desk Reference. In addition, byway of non-limiting example, a therapeutic compound may be believed tohave therapeutic activity on the basis of experience of a clinician,structure of the compound, structural activity relationship data, EC₅₀,assay data, IC₅₀ assay data, animal or clinical studies, or any otherbasis, or combination of such bases.

A therapeutically-active compound is a compound that has therapeuticactivity, including for example, the ability of a compound to induce aspecified response when administered to a subject or tested in vitro.Therapeutic activity includes treatment of a disease or condition,including both prophylactic and ameliorative treatment. Treatment of adisease or condition can include improvement of a disease or conditionby any amount, including prevention, amelioration, and elimination ofthe disease or condition. Therapeutic activity may be conducted againstany disease or condition, including in a preferred embodiment againstany disease or disorder that would benefit from dissociation of a tissueor mass of cells, for example. In order to determine therapeuticactivity any method by which therapeutic activity of a compound may beevaluated can be used. For example, both in vivo and in vitro methodscan be used, including for example, clinical evaluation, EC₅₀, and IC₅₀assays, and dose response curves.

In some embodiments, a pharmaceutical composition comprises a cationiclipid encapsulating a chimeric molecule embodied herein. In otherembodiments, the molecule comprises one or more sequences set forth asSEQ ID NOS: 1 to 95. In another embodiment, the one or more sequencescomprise SEQ ID NOS: 1 to 95 linked, fused, complexed with one or moreantimicrobial or membrane destabilizing domains.

Formulations, Administration:

The compositions embodied herein, are formulated for administration byany suitable method, for example, as described in Remington: The ScienceAnd Practice Of Pharmacy (21st ed., Lippincott Williams & Wilkins).Exemplary routes of administration include, but are not limited toparenteral, oral, subcutaneous, topical, intramuscular, transdermal,transmucosal, sublingual, intranasal, transvascular, subcutaneous,orbital, or combinations thereof.

Kits:

In yet another aspect, the invention provides kits for targeting nucleicacid sequences of cells and molecules associated with modulation of thetarget molecule. For example, the kits can be used to target any desirednucleic sequence and as such, have many applications.

In one embodiment, a kit comprises: (a) a cationic lipid, and a chimericmolecule or an encapsulated chimeric molecule, or a protein and aseparate polyanionic molecule, or any combinations thereof, and (b)instructions to administer to cells or an individual a therapeuticallyeffective amount of the composition. In some embodiments, the kit maycomprise pharmaceutically acceptable salts or solutions foradministering the composition. Optionally, the kit can further compriseinstructions for suitable operational parameters in the form of a labelor a separate insert. For example, the kit may have standardinstructions informing a physician or laboratory technician to prepare adose of chimeric molecule.

Optionally, the kit may further comprise a standard or controlinformation so that a patient sample can be compared with the controlinformation standard to determine if the test amount of chimericmolecule is a therapeutic amount consistent with for example, treatingdeafness in a patient.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1: Efficient Delivery of Genome Editing Proteins In Vitro and InVivo

Construction of Cas9, Cre, and TALE Fusion and sgRNA ExpressionPlasmids.

Sequences of all constructs used are listed below. All proteinconstructs were generated from previously reported plasmids for proteinof interest cloned into a pET29a expression plasmid.

Expression and Purification of S. pyogenes Cas9 and Other Proteins.

E. coli BL21 STAR (DE3) competent cells (Life Technologies) weretransformed with pMJ806 (Pattanayak, V. et al. Nat. Biotechnol. 31,839-843 (2013)) encoding the S. pyogenes Cas9 fused to an N-terminal10×His-tag (SEQ ID NO: 97)/maltose binding protein. The resultingexpression strain was inoculated in Luria-Bertani (LB) broth containing100 μg/mL of ampicillin at 37° C. overnight. The cells were diluted1:100 into the same growth medium and grown at 37° C. to OD₆₀₀=˜0.6. Theculture was incubated at 20° C. for 30 min, and isopropylβ-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce Cas9expression. After ˜16 h, the cells were collected by centrifugation at8,000 g and resuspended in lysis buffer (50 mMtris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 8.0, 1 M NaCl, 20%glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP)). The cells werelysed by sonication (1 sec pulse-on, 1 sec pulse-off for 15 min total at6 W output) and the soluble lysate was obtained by centrifugation at20,000 g for 30 min.

The cell lysate was incubated with His-Pur nickel-nitriloacetic acid(nickel-NTA) resin (Thermo Scientific) at 4° C. for 30 min to captureHis-tagged Cas9. The resin was transferred to a 20-mL column and washedwith 20 column volumes of lysis buffer. Cas9 was eluted in 50 mMTris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, 10 mM TCEP, and 300 mMimidazole, and concentrated by Amicon ultra centrifugal filter(Millipore, 100-kDa molecular weight cut-off) to ˜50 mg/mL. The 6×Histag (SEQ ID NO: 98) and maltose-binding protein were removed by TEVprotease treatment at 4° C. for 20 hours and captured by a secondNi-affinity purification step. The eluent, containing Cas9, was injectedinto a HiTrap SP HP column (GE Healthcare) in purification buffercontaining 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, and 10 mMTCEP. Cas9 was eluted with purification buffer containing a linear NaClgradient from 0.1 M to 1 M over five column volumes. The elutedfractions containing Cas9 were concentrated down to a concentration of200 μM as quantified by bicinchoninic acid assay (BCA) (PierceBiotechnology), snap-frozen in liquid nitrogen, and stored in aliquotsat −80° C. All other proteins were purified by this method but withoutTEV cleavage step and proteins containing (−30) GFP were purified byanion exchange using a Hi-Trap Q HP anion exchange column (GEHealthcare) using the same purification protocol.

In Vitro Transcription of sgRNAs.

Linear DNA fragments containing the T7 promoter binding site followed bythe 20-bp sgRNA target sequence were transcribed in vitro using the T7High Yield RNA Synthesis Kit (NEB) according to the manufacturer'sinstructions. In vitro transcribed RNA was precipitated with ethanol andpurified by gel electrophoresis on a Criterion 10% polyacrylamideTBE-Urea gel (Bio-Rad). Excised gel fragments were extracted in 420 μLof 300 mM NaCl overnight on a rocking surface at 4° C. Gel-purifiedsgRNA was precipitated with ethanol and redissolved in water and sgRNAconcentration was finally quantified by UV absorbance and snap-frozen at−80° C.

Plasmid Transfection.

Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies)according the manufacturer's protocol. For TALE activator plasmids, 300ng of DNA was transfected, and for the activator synergy experiments 60ng of each of five plasmids was pooled and transfected. For Cas9nuclease delivery experiments, linear DNA PCR products expressing sgRNAswere used in transfection experiments targeting genomic sites in CLTA,EMX, VEGF, and GFP (sgRNA GFP g1, GFP g3, GFP g5, and GFP g7 for nickasestudies). Linear DNA PCR products were generated using plasmidcontaining the U6 promoter as template and forward primers bearing theU6 promoter upstream sequence and reverse primers containing U6downstream sequence followed by the sgRNA sequence (20-bp sequenceunique to each target plus constant sgRNA backbone architecturesequence). sgRNAs expressed from linear DNA templates contained at leasttwo 5′ guanosines to match in vitro transcribed sgRNAs that requiredthese bases for T7 transcription. Primer sequences and PCR conditionsare referred to herein. For dCas9 activator experiments, 700 ng of Cas9or dCas9-VP64 plasmid DNA was co-transfected with 250 ng of theappropriate sgRNA expression plasmid. For activator synergy experiments50 ng of DNA from each of the six sgRNA was pooled and co-transfectedwith 700 ng of dCas9-VP64 plasmid.

Delivery of Transcription Factor Proteins Complexed with Cationic Lipidsin Cell Culture.

Briefly, cultured cells were plated in 48-well format (250 μL volume) inDulbecco's Modified Eagle's Media plus GLUTAMAX (Life Technologies,Carlsbad, Calif.) with 10% FBS (“full serum media”) and antibiotics at acell density necessary to reach ˜70% confluence the next day. Full serummedia was replaced with the same media but containing no antibiotics atleast one hour before delivery. Delivery of Cre and TALE proteins wasperformed by combining 1 nM to 1 μM protein (in 275 μL final volume)with 0.5-1.5 μL of commercially available cationic lipids in 25 μLOPTIMEM media (Life Technologies, Carlsbad, Calif.) according to themanufacturer's protocol for normal plasmid transfection, includingincubation time. For Cas9 delivery in vitro, transcribed sgRNA wasincubated with Cas9 protein for 5 min before complexing with thecationic lipid reagent. 25 μL lipid complexes in OPTIMEM media wereadded to cells and media was replaced 12-16 hours later fresh mediaunless otherwise noted. Cells were assayed for recombination 48 hoursafter delivery, for gene activation either 4 or 16 hours after delivery,and for gene modification 48 hours after delivery.

T7 Endonuclease I Assay to Detect Genomic Modifications.

U2OS-EGFP cells or HEK293T cells were transfected with Cas9 expressionand sgRNA expression plasmids or linear DNA PCR products as describedabove or treated with only Cas9 protein, only in vitro transcribedsgRNA, or only RNAiMAX. Genomic DNA was isolated from cells 2 days aftertransfection using the DNAdvance Kit (Agencourt) following themanufacturer's instructions. 200 ng of genomic DNA was used as templatein PCR reactions to amplify the targeted genomic loci with flankingsurvey primer pairs specified herein. PCR products were purified with aQIAquick PCR Purification Kit (Qiagen) and quantified with QUANT-IT™PICOGREEN dsDNA Kit (Life Technologies). 250 ng of purified PCR DNA wascombined with 2 μL of NEBuffer 2 (NEB) in a total volume of 19 μL anddenatured then re-annealed with thermocycling at 95° C. for 5 min, 95 to85° C. at 2° C./s; 85 to 20° C. at 0.2° C./s. The re-annealed DNA wasincubated with 1 μl of T7 Endonuclease I (10 U/μ1, NEB) at 37° C. for 15min. 10 μL of 50% glycerol was added to the T7 Endonuclease reaction and12 μL was analyzed on a 5% TBE 18-well Criterion PAGE gel (Bio-Rad)electrophoresed for 30 min at 200 V, then stained with 1×SYBR Gold (LifeTechnologies) for 30 min. Cas9-induced cleavage bands and the uncleavedband were visualized on an AlphaImager HP (Alpha Innotech) andquantified using ImageJ software (Schneider, C. A., et al. Nat. Methods9, 671-675 (2012)). The peak intensities of the cleaved bands weredivided by the total intensity of all bands (uncleaved+cleaved bands) todetermine the fraction cleaved which was used to estimate genemodification levels as previously described (Guilinger, J. P., et al.,Nat. Biotechnol. 32, 577-582 (2014)). For each sample, transfections andsubsequent modification measurements were performed in triplicate ondifferent days.

Stem Cell Culture and Delivery.

Mouse embryonic stem cell (ES) line Tau-GFP (courtesy of Dr. A. Edge,Massachusetts Eye & Ear Infirmary, Boston) containing a permanent GFPgene insertion was cultured in DMEM with 10% FBS (Gibco), 100 mM MEMnonessential amino acids (Gibco), 0.55 mM 2-mercaptoethanol, andleukemia inhibitory factor (1,000 units/ml; Chemicon). After 3 daysfloating spheres were formed that exhibited GFP fluorescence. Complexesof Cas9:sgRNA and Lipofectamine 2000 were added to the culturecontaining the floating spheres for 16 hours. After Cas9:sgRNAtreatment, the cells were cultured in the above media for 3 days. Thefloating spheres were treated with trypsin for 5 min then passed througha 70 μm filter to collect single cells. The cells were cultured onlaminin-coated slides in DMEM/F12 (1:1) supplemented with 1×N2, 1×B27,penicillin-streptomycin (100 μg/mL) and 10% FBS for two days beforelabeling. Immunohistochemistry was performed using an anti-GFP antibody(#ab13970, Abeam) to assess GFP expression. To quantify the number ofGFP-negative cells, the total number of GFP-positive and GFP-negativecells from three representative visual fields at 20× magnification werecounted, and the average efficiency was calculated. Three independentexperiments were performed for each condition.

Microinjection of Proteins to Mouse Inner Ear.

P1 floxP-tdTomato mice (The Jackson Laboratory) were used for(−30)GFP-Cre injection and P2 Atoh1-GFP mice (Dr. J Johnson,Southwestern Medical Center, University of Texas) were used forCas9:sgRNA injection. Animals were used under protocols approved by theMassachusetts Eye & Ear Infirmary ALCUC committee. Mice wereanesthetized by lowering their temperature on ice. Cochleostomies wereperformed by making an incision behind the ear to expose the cochlea.Glass micropipettes held by a micromanipulator were used to deliver thecomplex into the scala media, which allows access to inner ear haircells. For delivery of (−30)GFP-Cre, 3 μL of 45 μM protein was mixedwith 3 μL of either RNAiMAX or Lipofectamine 2000 and incubated at roomtemperature for 30 minutes prior to injection. Four mice were injectedper treatment group. For delivery of Cas9:sgRNA complexes, 1 μL of 200μM Cas9 protein was mixed with 2 μL of 100 sgRNA and incubated for 5minutes at room temperature before mixing with 3 μL of either RNAiMAX orLipofectamine 2000 and incubating for an additional 30 minutes prior toinjection. Three mice were injected per treatment group. The totaldelivery volume for every injection was 0.3 μL per cochlea and therelease was controlled by a micromanipulator at the speed of 32 nL/sec.

Immunohistochemistry and Quantification.

5-10 days after injection, the mice were sacrificed and cochlea wereharvested by standard protocols. For immunohistochemistry, antibodiesagainst hair-cell markers (Myo7a and Esp) and supporting cells (Sox2)were used following a previously described protocol (Sage, C. et al.Science 307, 1114-1118 (2005)). To quantify the number of tdTomatopositive cells after (−30)GFP-Cre or GFP negative cells after Cas9:sgRNAdelivery, the total number of outer hair cells were counted in a regionspanning 200 μm around the site of injection in the base turn of thecochlea. The efficiency of (−30)GFP-Cre-induced recombination orCas9:sgRNA-induced genome modification was calculated as the percentageof outer hair cells that expressed tdTomato or that lost GFP expression.

High-Throughput DNA Sequencing of Genome Modifications.

HEK293T cells were either transfected with Cas9 and sgRNA expressionplasmids or linear DNA PCR products or treated with 50 nM Cas9 protein,250 nM purified sgRNA, and cationic lipids as described earlier for Cas9protein delivery to U2OS-EGFP reporter cells. For plasmid-basedtransfection experiments, 700 ng of Cas9 expression plasmid plus 250 ngof sgRNA plasmid or 50 ng of a linear DNA PCR product expressing sgRNAfor targeting either the EMX1, CLTA2, or VEGF locus were transfectedwith Lipofectamine 2000 (Life Technologies) and cells were isolated 2days later. For protein delivery experiments in vivo, ˜˜30 mg of mousetissue was isolated from anesthetized mice and genomic DNA was extractedusing the Agencourt DNAAdvance Genomic DNA Isolation Kit (BeckmanCoulter). For cell culture experiments genomic DNA was isolated asdescribed above. 150 ng of genomic DNA was used as template to amplifyby PCR the on-target and off-target genomic sites with flanking HTSprimer pairs specified herein. Relative amounts of crude PCR productswere quantified by gel electrophoresis and samples treated withdifferent sgRNA pairs or Cas9 nuclease types were separately pooled inequimolar concentrations before purification with the QIAquick PCRPurification Kit (Qiagen). ˜150 ng of pooled DNA was electrophoresedusing a 5% TBE 18-well Criterion PAGE gel (BioRad) for 30 min at 200 Vand DNAs ˜125 bp to ˜300 bp in length were isolated and purified byQIAquick PCR Purification Kit (Qiagen). Purified DNA was amplified byPCR with primers containing sequencing adapters, purified, and sequencedon a MiSeq high-throughput DNA sequencer (Illumina) as previouslydescribed (Pattanayak, V. et al. Nat. Biotechnol. 31, 839-843 (2013)).

Quantification of Cas9 Protein Uptake.

Alexa Fluor 647 C2 Maleimide (Life Technologies, Carlsbad Calif.) wasused to fluorescently label Cas9 protein on surface cysteines. A 10 mMstock solution of Alexa647 was prepared in anhydrous DMSO. In a 0.4 mLreaction, 10 nmol of purified Cas9 protein and 200 nmol of Alexa647maleimide were combined in buffer conditions used for Cas9 proteinstorage. The labeling reaction was incubated at 4° C. for 16 hours. Atthe end of the reaction, excess unconjugated Alexa647 was removed byre-purifying the labeled Cas9 protein by cation exchange chromatographyas described above. To measure the amount of protein delivered intotreated cells, 20,000 cells were plated in the wells of a 48-well plate1 day prior to treatment. On the day of treatment, 50 nM ofAlexa647-labeled Cas9 (Cas9-Alexa647) and 100 nM of EGFP1 sgRNA wereprepared for delivery using 0.8 μL of Lipofectamine 2000 as describedabove, and applied to the cells. After 4 hours, Cas9-Alexa647:sgRNALipofectamine-containing media was removed, and cells were washed threetimes with 500 μL of PBS containing 20 U/mL heparin.

The cells were trypsinized and prepared for counting and flow cytometryas described above. Cas9-Alexa647 uptake was measured by flow cytometry,while 10,000 cells of the treated population were transferred to ablack, flat-bottomed, opaque 96-well plate. Standard curves ofCas9-Alexa647 were prepared by complexing 50 pmol of the Cas9-Alexa647protein with Lipofectamine 2000 exactly as described for Cas9-Alexa647delivery, followed by serial 2-fold dilutions in DMEM with 10% FBScontaining 10,000 U2OS cells per well in the 96-well plate. The effectof U2OS cells or complexation with Lipofectamine 2000 on Alexa647fluorescence was determined by preparing three additional Cas9-Alexa647standard curves: (i) with Lipofectamine 2000 in media lacking U2OScells, (ii) without Lipofectamine 2000 in media containing U2OS cells,and (iii) without Lipofectamine 2000 in media lacking U2OS cells.

Data Analysis.

Illumina sequencing reads were filtered and parsed with scripts writtenin Unix Bash. DNA sequences will be deposited in NCBI's Sequencing ReadsArchive (SRA) and source code can be found in Supplementary Software.Sample sizes for sequencing experiments were maximized (within practicalexperimental considerations) to ensure greatest power to detect effects.Statistical analyses for Cas9-modified genomic sites (Table 2) wereperformed as previously described (Sander, J. D. et al. Nucleic AcidsRes. 41, e181 (2013)).

Amino acid sequences of proteins used in this study

(+36)GFP-Cre-6xHis (SEQ ID NO: 1):MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMERDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSET GAMVRLLEDGDGGSHHHHHH(−7)GFP-Cre-6xHis (SEQ ID NO: 2):MGASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSET GAMVRLLEDGDGGSHHHHHH(−20)GFP-Cre-6xHis (SEQ ID NO: 3):MGASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSET GAMVRLLEDGDGGSHHHHHH(−30)GFP-Cre-6xHis (SEQ ID NO: 4):MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSET GAMVRLLEDGDGGSHHHHHHCre-6xHis (SEQ ID NO: 5):MASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHH HHHCas9 (SEQ ID NO: 6): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDCas9-6xHis (SEQ ID NO: 7):MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHH NLS-Cas9-6xHis (SEQ ID NO: 8):MPKKKRKVMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHH Cas9-NLS-6xHis (SEQ ID NO: 9):MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVMDKHHHHHH(+36)dGFP-NLS-Cas9-6xHis (Y67S) (SEQ ID NO: 10):MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTSGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHH HHH(−30)dGFP-NLS-Cas9-6xHis (Y67S) (SEQ ID NO: 11):MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTSGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVICKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHH HHHHdCas9-VP64-6xHis (D10A and H840A) (SEQ ID NO: 12):MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEH1ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAGGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS DALDDFDLDMLHHHHHHCas9 nickase (D10A) (SEQ ID NO: 13):MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHH (−30)GFP-Cre-6xHis (SEQ ID NO: 14):ATGGGTGCTAGCAAAGGTGAAGAGCTGTTTGACGGTGTAGTACCGATCTTAGTGGAATTAGACGGCGACGTGAACGGTCACGAATTTAGCGTGCGCGGCGAGGGCGAAGGTGACGCTACCGAGGGTGAATTGACCCTGAAGTTTATTTGCACAACAGGCGAATTACCCGTTCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTGATTACCCAGATCATATGGATCAACACGATTTTTTCAAATCAGCCATGCCTGAAGGATATGTTCAAGAGCGTACAATCAGCTTCAAGGACGATGGCACCTATAAAACGCGTGCGGAAGTGAAATTTGAAGGCGACACATTAGTAAACCGTATCGAACTGAAAGGTATCGACTTCAAAGAAGACGGCAACATTTTAGGCCATAAGCTGGAATATAACTTTAATTCTCATGACGTGTATATTACGGCCGATAAACAGGAAAACGGTATCAAGGCAGAATTTGAAATTCGCCATAACGTGGAGGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGCCGATTGGTGATGGGCCTGTACTGTTACCTGACGATCACTACCTGAGCACGGAGTCAGCCCTGAGCAAAGATCCGAACGAAGACCGCGATCACATGGTTCTGTTAGAATTCGTGACCGCTGCAGGCATTGATCATGGAATGGACGAGCTGTACAAGACCGGTGGTAGCGGTGGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGGCAGCGGCGGTACCGCGAGCAATTTACTGACCGTACACCAAAATTTGCCTGCATTGCCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGTATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGACGGCGGATCCCATCACCA CCACCATCACCre-6xHis (SEQ ID NO: 15):ATGGCGAGCAATTTACTGACCGTACACCAAAATTTGCCTGCATTGCCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGTATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGACGGCGGATCCCATCACCAC CACCATCAC(−30)dGFP-NLS-Cas9-6xHis (SEQ ID NO: 16):ATGGGTGCTAGCAAAGGTGAAGAGCTGTTTGACGGTGTAGTACCGATCTTAGTGGAATTAGACGGCGACGTGAACGGTCACGAATTTAGCGTGCGCGGCGAGGGCGAAGGTGACGCTACCGAGGGTGAATTGACCCTGAAGTTTATTTGCACAACAGGCGAATTACCCGTTCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTGATTACCCAGATCATATGGATCAACACGATTTTTTCAAATCAGCCATGCCTGAAGGATATGTTCAAGAGCGTACAATCAGCTTCAAGGACGATGGCACCTATAAAACGCGTGCGGAAGTGAAATTTGAAGGCGACACATTAGTAAACCGTATCGAACTGAAAGGTATCGACTTCAAAGAAGACGGCAACATTTTAGGCCATAAGCTGGAATATAACTTTAATTCTCATGACGTGTATATTACGGCCGATAAACAGGAAAACGGTATCAAGGCAGAATTTGAAATTCGCCATAACGTGGAGGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGCCGATTGGTGATGGGCCTGTACTGTTACCTGACGATCACTACCTGAGCACGGAGTCAGCCCTGAGCAAAGATCCGAACGAAGACCGCGATCACATGGTTCTGTTAGAATTCGTGACCGCTGCAGGCATTGATCATGGAATGGACGAGCTGTACAAGACCGGTGGTAGCGGTGGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGGCAGCGGCGGTACCGCGCTCGCGCTGCCCAAGAAGAAGAGGAAGGTGATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACCATCACCAC CACCATCAC(+36)dGFP-NLS-Cas9 (SEQ ID NO: 17):ATGGGTGCTAGCAAAGGTGAACGTCTGTTTCGTGGTAAAGTACCGATCTTAGTGGAATTAAAGGGCGACGTGAACGGTCATAAATTTAGCGTGCGCGGCAAAGGCAAAGGTGACGCTACCCGTGGTAAATTGACCCTGAAGTTTATTTGCACAACAGGCAAATTACCCGTTCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTCGTTACCCTAAACATATGAAACGTCACGATTTTTTCAAATCAGCCATGCCTAAAGGATATGTTCAAGAGCGTACAATCAGCTTCAAGAAGGATGGCAAATATAAAACGCGTGCGGAAGTGAAATTTGAAGGCCGCACATTAGTAAATCGTATCAAACTGAAAGGTCGTGACTTCAAAGAAAAAGGCAACATTTTAGGCCATAAACTGCGTTATAACTTTAATTCTCATAAGGTGTATATTACGGCCGATAAACGCAAGAATGGTATCAAGGCAAAATTCAAAATTCGCCATAACGTGAAAGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGCCGATTGGTCGCGGGCCTGTACTGTTACCTCGCAACCACTACCTGAGCACCCGTTCTAAACTGAGCAAAGATCCGAAAGAAAAACGCGATCACATGGTTCTGTTAGAATTCGTGACCGCTGCAGGCATTAAGCACGGACGCGACGAACGCTACAAGACCGGTGGTAGCGGTGGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGGCAGCGGCGGTACCGCGCTCGCGCTGCCCAAGAAGAAGAGGAAGGTGATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACCATCACCAC CACCATCACCas9-NLS-6xHis (SEQ ID NO: 18):ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACCCCAAGAAGAAGAGGAAGGTGATGGATAAGCATCACCACCACCATC ACdCas9-VP64-6xHis (SEQ ID NO: 19):ATGGATAAGAAATACTCAATAGGCTTAGCTATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATGCCATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACGGTTCTCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGAGGCGGTGGAAGCGGGCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCTCGATATGTTACATCACCACCACCATCA C

Example 2: Delivery of Cas9:gRNA in Adult Cochlea In Vivo

To study if Cas9 protein with gRNA can be delivered to adult cochlea invivo, Cas9:gRNA-GFP complexed with lipofectamine2000 was microinjectedin 1-month-old Atoh1-GFP mouse cochlea. Two weeks after injectiondisappearance of GFP signals in some outer hair cells (FIG. 1B, OHC inbrackets) was observed whereas in control saline injected inner ear allOHCs have GFP signal (FIGS. 1A, 1B). OHCs without GFP could still beco-labeled with DAPI and Myo7a, a demonstration of survival of OHCs.

Cas9:gRNA Mediated Genome Editing in Pmca2 Mutant Mouse with Restorationof Hearing:

Mutation in Pmca2 gene (2765C→T) causes hereditary hearing loss intransgenic mouse (Oblivion, Obl) (Spiden et al., 2008; Street,McKee-Johnson, Fonseca, Tempel, & Noben-Trauth, 1998). To study ifCas9:gRNA can be used to disrupt the mutation to restore hearing, a setof gRNAs were designed, that target the mutation for Cas9:gRNA injectioninto neonatal Obl heterozygous (Pmca2^(Obl/+)) mouse inner ear atpostnatal day 1 (P1) (FIG. 2; SEQ ID NOS: 21-32). A hearing study (ABRand DPOAE) was performed on the injected and uninjected inner ears, one,two and three months after injection.

In the uninjected cochlea, the hearing test showed profound hearing lossand the lack of DPOAE at the highest sound level used (90 dB) across allfrequencies at one month of age, and hearing loss persisted throughoutthe rest of study (FIG. 3). In the injected mouse cochlea, drastichearing improvement was observed, shown by ABR and DPOAE in allfrequencies except for the highest frequency of 45.25 kHz. Hearingrestoration was maintained in two and three month old cochleas injectedwith Cas9:gRNA (FIG. 3). Of all the gRNAs that targeted the mutation,only one (2.4-mut) showed hearing recovery, strongly supporting theimportance in selecting gRNA with the specific effect (FIG. 4). Tofurther evaluate the specificity of the study, gRNAs were designedagainst wildtype Pmca2 gene for inner ear injection (FIG. 2; SEQ ID NOS:21-32). With wildtype gRNAs hearing improvement was not detected in thePmca2^(Obl/+) cochlea injected animals. T7EI endonuclease assay wasperformed and showed that in vitro Cas9:gRNA cleaved mutant DNA (FIG. 5)at 125 nM. High-throughput sequencing (HTS) was performed to confirmthat the strategy induces indels (insertions and deletions) in theinjected inner ear, to provide molecular proof of genome editing.

To further correlate hearing recovery with hair cell status, injectedand control inner ears were examined. By immunolabeling it was foundthat a majority of outer hair cells survived after injection whereas incontrol inner ear a majority of outer hair cells died especially in thebase to middle turn (FIGS. 6A, 6B). In comparison to control, theinjected inner ear maintained a large number neurites of spiral ganglionneurons (FIGS. 7A, 7B). Thus Cas9:gRNA mediated genome editing leads toouter hair cell survival and maintain their connection with neurons,resulting hearing recovery.

As mutations in PMCA2 gene are involved in human genetic deafness(Schultz, J. M. et al., 2005, N Engl J Med. 352(15), 1557-1564) thisapproach would lead to intervention in those patients. While this studyis focused on the disruption of dominant mutation, it is highly feasiblethat a similar approach can be applied to recessive mutations whenefficient for homology-directed repair (HDR) is improved.

An additional mechanism for hearing recovery in the injected inner earcould be due to interference of Cas9:gRNA that blocks of Pmca2 mutanttranscription. Such interference has been previously reported (LRussa &Qi, 2015, Mol Cell Biol. doi.org/10.1128/MCB.00512-15). Interference byCas9:gRNA to disrupt mutant gene that leads to functional recovery ofhearing is a new mechanism that can be applied to other dominant hearingloss and genetic diseases in general.

Hearing Restoration in Tmc1 Beethoven (Bth) Genetic Hearing Loss MouseModel:

To further study the generality of the Cas9:gRNA approach in restoringhearing, a Tmc1 mouse mutant (Bth) was used due to a dominant mutation(1235T→A that leads to M412K) (Vreugde, S., et al., (2002). Nat Genet,30(3), 257-258), using the same procedure as outlined in Pmca2 study. Aseries of gRNA against the mutation as well as wildtype Tmc1 gene weredesigned. Tmc1 heterozygous mouse inner ear were injected with Cas9:gRNAat P1 and their hearing was assessed at 4 weeks of age. The uninjectedinner ears served as control.

By ABR, a generally improved hearing was found across all frequencieswith significant improvements at the frequencies of 11.32, 16 and 22.64kHz. Over 20 dB improvements were seen in the latter two frequencies.For the Bth mice DPOAE were present. The injected ears had DPOAE similarto the uninjected inner ears with a slight elevation from 8 to 16 kHz(FIG. 8). In the Bth mice, hearing loss was largely due to dysfunctionalinner hair cells, in contrast to Pmca2 mice where outer hair cells areaffected. Combining Pmca2 and Tmc1 studies, it was demonstrated thatthis approach is suitable to both inner and outer hair cell relatedhearing loss. In human the same Tmc1 mutation (M412K) has been reportedin a multi-generation family (Zhao Y. et al. PLoS ONE, 9(5), e97064.http://doi.org/10.1371/journal.pone.0097064). This study has potentialto be developed as a treatment to be applied to those patients as a newtherapy.

Example 3: Rescue of Hearing Loss by Cas9/gRNA Delivery In Vivo andCRISPR Mediated Gene Editing in a Genetic Deaf Mouse Model

To use liposomal formulation that complexes Cas9 with gRNA for CRISPRmediated gene editing as a potential treatment for genetic deafness, arescue effect on Pmca2 deafness mouse mutant was studied. Pmca2 is aplasma membrane Ca²⁺ pump that is highly expressed in the inner ear haircells, with the function that actively pumps out Ca²⁺ that enters haircells during signal mechanoelectrical transduction during hearing andvestibular function. PMCA2 mutation has been shown to increase hearingloss severity human (M Schultz et al., “Modification of Human HearingLoss by Plasma-Membrane Calcium Pump PMCA2,” N Engl J Med 352, no. 15(Apr. 14, 2005): 1557-64, doi:10.1056/NEJMoa043899). In the mouse mutant(Oblivion) with a point mutation (S877F), severe to profound (i.e.complete) hearing loss is observed in heterozygous and homozygous mice(Spiden et al., PLoS Genetics 4, e1000238-e1000238.2008). This mousemutant thus serves as an excellent model to determine if the Cas9/gRNAapproach can be used to disrupt the Pmca2 mutation in heterozygous micefor hearing recovery, with implication to reduce hearing loss in human.

To study the hearing rescue effect, 12 guide RNAs were designed, 4 ofwhich targeted the mutation. Following lipofectamine 2000 formulationthat complexes Cas9 with each gRNA, the complex was injected intopostnatal day 3 (P3) mouse cochleas. Both mouse mutants and wildtypecontrol mice were injected. For each mouse right ear was injected andthe left ear was uninjected. Three weeks or four weeks after injection,acoustic Auditory brainstem response (ABR) and distortion productoptoacoustic emissions (DPOAE) tests were performed.

For ABR and DPOAE tests, injected mice of either sex were anesthetizedwith xylazine (10 mg/kg, i.p.) and ketamine (100 mg/kg, i.p.). ABR andDPOAE were performed as previously described (Huang et al., 2013). ABRmeasures the auditory pathway from hair cells to brain; whereas DPOAEmeasures primarily outer hair cell function. By their combination itcould be inferred if the hearing defects are of hair cells or centralpathway deficiency.

At three weeks after injection, in the heterozygous Pmca2 mice,uninjected inner ears had profound hearing loss as shown by ABR andDPOAE. In the Cas9/gRNA-Pmca-2.4 (with the guide RNA 2.4) injected ears,significant hearing recovery in frequencies of 16, 22.64, 32 and 45.24kHz by ABR was observed. By DPOAE, significant recovery in frequenciesfrom 16 to 45.24 kHz was detected in the Cas9/gRNA-Pmca-2.4 injectedinner ear, corresponding to ABR recovery. Recovery of DPOAE is anindication of restoration of hair cell function. To study long-termeffect of hearing recovery, a hearing study was performed four weeksafter injection and observed similar hearing recovery. The hearing studyis continued at 6, 12 and 26 weeks after injection. In addition to theuninjected control ears, Pmca2 heterozygous mice injected with Cas9complexed with other Pmca2 guide RNAs were also studied. No hearingrecover was detected either by ABR or DPOAE was detected (data notshown). Thus guide RNA Pmca2-2.4 complexed with Cas9 induced sequencespecific gene editing of Pmca2 mutation, leading to significantimprovement of hearing.

To study potential toxicity associated with Cas9/gRNA delivery,Cas9/gRNA-Pmca-2.4 was injected into P3 wildtype (WT) mice and performedhearing study 3 weeks after injection. Slight elevation was observed inABR and DPOAE at the highest frequency (45.24 kHz), but not in any otherfrequencies. Thus Cas9/gRNA-Pmca-2.4 complex does not cause additionaldamage to healthy hair cells or inner ear function. All together thestudy demonstrates that Cas9/gRNA that targets Pmca2 mutation in haircells restores hearing in otherwise complete deaf mouse mutants. Thesimilar strategy thus can be applied to human deaf patients with Pmca2mutations to improve or restore hearing.

The hearing recovery in the Cas9/gRNA-Pmca-2.4 injected ear was notuniform across all frequencies (e.g. no recovery in 8 kHz). Further therecovery was uneven as better recovery was seen at the highest frequencyof 45.24 kHz. This is likely due to the surgical procedure used onlyallowed for access to primarily the base of the cochlea, which isresponsible for high-frequency hearing. The lack of recovery at the lowfrequency is likely due to the insufficient diffusion of Cas9/gRNAcomplex to the apical region of the cochlea. To test the hypothesis,additional experiments by multiple injections were performed in thePmca2 mice over 6 days. By four weeks much greater hearing recovery wasobserved (40 dB) covering a majority of frequencies from 16 to 45.24 kHz(FIG. 9). The mouse inner ear is extremely small in size, about 1/50thof the size of human inner ear. While the mouse inner ear presents asurgical challenge in protein delivery, it is anticipated that in humaninner ear the delivery would be considerably easier. Thus multipleinjections result in greatly improved hearing restoration across mostfrequencies.

One of the most important applications of the technology is the abilityto deliver the Cas9/gRNA complex in mature mammalian inner ear. Thefirst set of experiments were conducted and showed that when injectedinto P9 mouse cochlea, a similar hearing rescue effect was observed(data not shown).

The work demonstrates the utility of direct Cas9/gRNA delivery intomammalian inner ear hair cells in vivo in disruption of mutations thatleads to functional recovery of hearing. As 20% of genetic deafness isdue to dominant mutations, this method can be tailored to target thosemutations to restore hearing.

The most common form of deafness is recessive, for which repair ofmutations will be needed for hearing restoration. One of the most commonforms of deafness in human is age-related hearing loss (ARHL) orpresbycusis, affecting over hundreds of millions of people worldwide.While the major mechanisms underlying ARHL is unknown, it is likely thatgenes will be identified with mutations or polymorphisms that make haircells vulnerable to aging. Under this condition, the Cas9/gRNA could beapplied to disrupt or repair the mutations/polymorphisms, to restore orslow down the progression of hearing loss. While the method currentlytargets hair cells, modifications will be made so that the method can beused to target inner ear cell types such as supporting cells, strialvascular and neurons, in which similar gene editing can be achieved forfunctional recovery of hearing. Finally many recessive genetic deafnessis congenital, by the time of birth, simple gene editing may not besufficient to restore cell function or hearing due to degeneration ofthe cell types. However it is possible to combine regeneration of thecell types with gene editing, to produce new cells while correctingmutations. These combinations can be applied to restore hearing inpatients suffering from hearing loss due to different causes.

Example 4: Protein Delivery into the Inner Ear

The goal of this study was to use protein-mediated delivery system todeliver the biological proteins directly inner ear cell types withfunctional consequences. With this method, the proteins delivered havespecific functions, and the effect is transient. Further, delivery ofnative protein lessens any potential immune response.

As cell surface is cationic (positively charged), it has beendemonstrated that, by changing amino acids, GFP (green fluorescentprotein) can be modified to be highly charged (both positively andnegatively, named supercharged protein), which enables the superchargedGFP to enter cells. As a result, the super-charged GFP (s-GFP) can beused as vehicle to link and deliver other proteins into cells withbiological effect in vitro and in vivo. This has been demonstrated inmouse retina in which s-GFP carrying Cre recombinase (s-GFP-Cre) wasdelivered that resulted in expression of reporter gene in retinal cells.The s-GFP used as (−30)-GFP, a negatively charged GFP protein.

The use of (−30)-GFP protein and a new positively charged (+36)-GFPprotein to carry Cre, (−30)-GFP-Cre and (+36)-GFP-Cre, was tested forinjection into mouse cochlea in vivo. The mouse used wasRosa-tdTomato^(f/f) in which functional Cre activity in the nucleiresults in cells being labeled with tdT (red). It was shown that both(−30)-GFP-Cre and (+36)-GFP-Cre injections led to tdT expression in thecochlear hair cells. Near the injection site (base turn of cochlea),˜30% of hair cells became tdT-labeled (FIGS. 10A, 10B), The studydemonstrated that supercharged GFP proteins can be used to deliveryprotein directly into inner ear hair cells with biological effect innuclei.

It was next tested whether Aurein, an antimicrobial peptide, incombination with s-GFP, can serve as an effective carrier to deliverfunctional proteins into mouse inner ear cell types with highefficiency. (+36)-GFP-aurein-Cre was injected with differentconcentrations into P3 Rosa-tdT mouse inner ear by cochleostomy in vivo.The tissues were harvested 5 days later for immunolabeling to identifytdT⁺ inner ear cell types. Overall it was found that cochlear hair cellswere primarily labeled with tdT, with additional tdT labeling insupporting cells and in spiral ganglion neurons. At low concentrationsof 5 μM, few IHC and some supporting cells (SC) were tdT⁺. At 22.5 μM,58% of OHC and 75% of IHC, as well as 8% of SC were tdT⁺. At the highest50 μM, 96% of OHC, 88% of IHC and 19% of SC were tdT⁺. Some spiralganglion neurons were also tdT⁺ at 22.5 and 50 μM (FIGS. 11A-11E andFIG. 12). There was slight cell toxicity associated with the highestconcentration of 50 μM. It was found that 20% of IHC loss 8% of OHC inthe injected animals, whereas at lower concentrations of 5 and 22.5 μMno cell loss was detected.

The study demonstrated that functional proteins including nuclearproteins can be conjugated with supercharged protein (+36)GFP fused withaurein. The protein complex was directly injected into mammalian innerear that leads to uptake by a wide range of inner ear cell types. Theprotein delivered was properly localized in the nuclei and had thespecific biological function in cleaving floxP sites on the DNAsequence, to activate tdTomato in the inner ear cells with highefficiency.

The delivery system can be used to study protein functions, inner earregeneration for hearing recovery, hearing protection, and gene editingto correct genetic deafness by genome-editing proteins.

Example 5: Characterization of a Peptide that Enhances Endosomal Escapeof Delivered Proteins In Vitro and In Vivo

In this study, aurein 1.2 (GLFDIIKKIAESF; SEQ ID NO: 34) was discoveredas a peptide that enhances the endosomal escape of a variety of cargofused to +36 GFP. The structure-function relationships were elucidatedwithin aurein 1.2 using alanine scanning and mutational analysis.Results from three independent delivery assays confirmed that treatmentof mammalian cells with cargo proteins fused to aurein 1.2-+36 GFPresult in more efficient cytosolic delivery than the same proteins fusedto +36 GFP alone. The ability of aurein 1.2 to enhance non-endosomalprotein delivery in vivo is also described. Cre recombinase enzyme wasdelivered into hair cells in the cochlea (inner ear) of live mice withmuch greater (>20-fold) potency when fused with aurein 1.2 than in theabsence of the peptide. These results together provide a simplemolecular strategy for enhancing the cytosolic delivery of proteins incell culture and in vivo that is genetically encoded, localized to cargomolecules, and does not require global treatment with cytotoxic smallmolecules.

Materials and Methods

Expression and Purification of Proteins.

E. coli BL21 STAR (DE3) competent cells (Life Technologies) weretransformed with pET29a expression plasmids. Colonies from the resultingexpression strain was directly inoculated in 1 L of Luria-Bertani (LB)broth containing 100 μg/mL of ampicillin at 37° C. to OD₆₀₀=˜1.0.Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM toinduce expression and the culture was moved to 20° C. After ˜16 h, thecells were collected by centrifugation at 6,000 g and resuspended inlysis buffer (Phosphate buffered saline (PBS) with 1 M NaCl). The cellswere lysed by sonication (1 sec pulse-on, 1 sec pulse-off for 6 min,twice, at 6 W output) and the soluble lysate was obtained bycentrifugation at 10,000 g for 30 min.

The cell lysate was incubated with His-Pur nickel-nitriloacetic acid(Ni-NTA) resin (Thermo Scientific) at 4° C. for 45 min to captureHis-tagged protein. The resin was transferred to a 20-mL column andwashed with 20 column volumes of lysis buffer plus 50 mM imidazole.Protein was eluted in lysis buffer with 500 mM imidazole, andconcentrated by Amicon ultra centrifugal filter (Millipore, 30-kDamolecular weight cut-off) to ˜50 mg/mL. The eluent was injected into a 1mL HITRAP SP HP column (GE Healthcare) after dilution into PBS (5-fold).Protein was eluted with PBS containing a linear NaCl gradient from 0.1 Mto 1 M over five column volumes. The eluted fractions containing proteinwere concentrated to 50 μM as quantified by absorbance at 488 nmassuming an extinction coefficient of 8.33×10⁴ M⁻¹cm⁻¹ as previouslydetermined (McNaughton, B. R.; et al. Proceedings of the NationalAcademy of Sciences 2009, 106, 6111), snap-frozen in liquid nitrogen,and stored in aliquots at −80° C.

Cell Culture.

All cells were cultured in Dulbecco's modification of Eagle's medium(DMEM w/glutamine, Gibco) with 10% fetal bovine serum (FBS, Gibco), 5I.U. penicillin, and 5 g/mL streptamycin. All cells were cultured at 37°C. with 5% CO₂.

Peptide Synthesis.

Peptides were ordered from ChinaPeptides Co., LTD, each 4 mg,purity>90%. HPLC and MALDI data were provided with lyophilized peptides.Peptides were resuspended in DMSO to a final concentration of 10 mM.

Sortase Conjugation.

All reactions were performed in 100 mM Tris buffer (pH 7.5) with 5 mMCaCl₂ and 1M NaCl. For peptide conjugation to the N-terminus ofGGG-+36-GFP, 20 μM of protein with N-terminal Gly-Gly-Gly was incubatedwith 400 μM of peptide with C-terminal LPETGG (SEQ ID NO: 89) and 1 μMeSrtA for 2 h at room temperature in a 50 μL reaction. The unreactedpeptides were removed through spin filtration with an Amicon Ultra-0.5Centrifugal Filter Unit (Millipore, 30-kDa molecular weight cut-off).The reaction mixture was washed twice with 500 μL of buffer each time toa final concentration of 50 μL. Conjugation efficiency was determinedthrough LC-MS (Agilent 6220 ESI-TOF) using protein deconvolution throughMaxEnt (Waters) by comparing relative peak intensities.

For conjugation of GGGK^(Dex) (SEQ ID NO: 99) to +36-GFP-LPETG-His₆(“LPETG-His₆” disclosed as SEQ ID NO: 100), 10 μM of protein wasincubated with 400 μM of peptide and 2 μM eSrtA at room temperature. Thereaction was quenched with 10 mM ethylenediaminetetraacetic acid (EDTA)after 2 h. For aurein 1.2-+36-GFP-LPETG-His₆ (“LPETG-His₆” disclosed asSEQ ID NO: 100), a N-terminal His₆-ENLYFQ (SEQ ID NO: 101) was added toprevent sortase reaction with the N-terminal glycine of aurein 1.2. TheN-terminal tag was removed with 200 μM TEV protease at 4° C. for 16 h torelease the native N-terminal sequence of aurein 1.2-+36-GFP. Successfulconjugation of GGGK^(Dex) (SEQ ID NO: 99) removes the C-terminal His₆tag (SEQ ID NO: 98) and allows for purification through reverse Ni-NTAcolumn. Unreacted protein binds to the Ni-NTA, and the unbound proteinwas collected and concentrated as described above.

Plasmid Transfection.

Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies)according the manufacturer's protocol.

Synthesis of Dexamethasone-21 Thiopropionic Acid (SDex).

Synthesis of dexamethasone-21-mesylate was performed as previouslydescribed (Simons, S. S.; et al. J Org Chem 1980, 45, 3084; Dunkerton,L. V.; et al. Steroids 1982, 39, 1). 2 g of dexamethasone stirring in 38mL anhydrous pyridine under nitrogen was reacted with 467.2 mgmethanesulfonyl chloride (1.2 eq.) on ice for 1 h, after which another311 methanesulfonmethanesulfonyl chloride was added and allowed to reactovernight (16 h) on ice. Next, the reaction was added to 800 mL of icewater and Dexamethasone-21-Mesylate (Dex-21-OMs) formed a whiteprecipitate. The slurry was filtered and the precipitate washed with 800mL of ice water, dried under high vacuum overnight and quantified byLC-MS (m/z 471.19 Da, 83% yield).

Dexamethasone-21-thiopopionic acid (S^(Dex)) was prepared as previouslydescribed (Kwon, Y. U.; Kodadek, T. J Am Chem Soc 2007, 129, 1508). 2.05g of Dex-21-OMs was added to 2 eq. thiopropionic acid and 4 eq.triethylamine stirring in anhydrous acetone at room temperatureovernight. The following morning, the reaction was added to 800 mL ofice water and acidified with 1N HCl until S^(Dex), visible as anoff-white solid, precipitation was complete. The mixture was filtered,washed with 800 mL ice cold water acidified to pH 1 with HCl, driedunder high vacuum overnight and analyzed by LC-MS (m/z 481.21 Da, 63%yield).

Synthesis and Purification of GGGK^(Dex) (SEQ ID NO: 99).

GGGK^(Dex) (SEQ ID NO: 99) was synthesized on Fmoc-Lys (Mtt)-Wang resin(BACHEM, D-2565) using microwave acceleratin (MARS, CEM). Couplingreactions were performed using 5 equivalents of Fmoc-Gly-OH(Novabiochem, 29022-11-5), 5 equivalents of PyClock (Novabiochem,893413-42-8) and 10 equivalents of diisopropylethylamine (DIEA) inN-methylpyrrolidone (NMP). Fmoc groups were removed using 25% piperidinein NMP (efficiency quantified; ε₂₉₉=6234 M⁻¹cm⁻¹ in acetonitrile) andMtt groups were removed by incubating the Fmoc-GGGK(Mtt)-resin (SEQ IDNO: 99) with 2% trifluoroacetic acid (TFA) in dichloromethane (DCM) for20 min, after which the resin was washed with 2% TFA in DCM until thecharacteristic yellow color emitting from the Mttcation subsided. AfterMtt removal, S^(Dex)-COOH (Dex-21-thiopropinonic acid) was coupled tothe Nε of the lysine side-chain by incubating the Fmoc-GGGK-resin (SEQID NO: 99) with 2.5 eq. S^(Dex)-COOH, 2.5 eq. HATU, 2.5 eq. HOAt, 5 eq.DIEA and 5 eq. 2,6-lutidine in 2.5 mL NMP overnight, at roomtemperature, on an orbital shaker. After S^(Dex)-labeling,Fmoc-GGGK^(Dex)-resin (SEQ ID NO: 99) was washed thoroughly with NMP andDCM, the N-terminal Fmoc was removed using 25% piperidine in NMP, andcrude peptides were dissociated from the resin by incubating theGGGK^(Dex)-resin (SEQ ID NO: 99) in a cleavage cocktail composed of81.5% trifluoroacetic acid (TFA), 5% thioanisole, 5% phenol, 5% water,2.5% ethanedithiol and 1% triisopropylsilane for 30 min at 38° C. Crudepeptides were precipitated in 40 mL cold diethyl ether, resuspended inwater, lyophilized and purified via reverse phase high-pressure liquidchromatography (HPLC) using a linear gradient of acetonitrile and waterwith 0.1% TFA across a C18 (VYDAC, 250 mm×10 mm ID) column. Purifiedpeptides were lyophilized and stored at 4° C. Polypeptide identity wasconfirmed by mass spectrometry on a Waters QToF LC-MS, and purity wasmeasured by analytical reverse-phase HPLC (Shimadzu Instruments) using aC18 column (Poroshell 120 SB-C18, 2.7 μm, 100 mm×3 mm ID, Agilent).

Image Processing for Primary Screen.

BSR.LNL.tdTomato cells were plated at 10,000 cells per well in black384-well plates (Aurora Biotechnologies). Cells were treated with Crefusion proteins diluted in serum-free DMEM 24 h after plating andincubated for 4 h at 37° C. Following incubation, the cells were washedthree times with PBS+20 U/mL heparin. The cells were incubated a further48 h in serum-containing media. Cells were fixed in 3% paraformaldehydeand stained with Hoescht 33342 nuclear dye. Images were acquired on anImageXpress Micro automated microscope (Molecular Devices) using a 4×objective (binning 2, gain 2), with laser- and image-based focusing(offset −130 μm, range ±50 μm, step 25 μm). Images were exposed for 10ms in the DAPI channel (Hoechst) and 500 ms in the dsRed channel(tdTomato). Image analysis was performed using the cell-scoring moduleof MetaXpress software (Molecular Devices). All nuclei were detectedwith a minimum width of 1 pixel, maximum width of 3 pixels, and anintensity of 200 gray levels above background. Positive cells wereevaluated for uniform signal in the dsRed channel (minimum width of 5pixels, maximum width of 30 pixels, intensity>200 gray levels abovebackground, 10 μm minimum stained area). In total, nine images werecaptured and analyzed per well, and 16 wells were treated with the samefusion protein. The primary screen was completed in biologicaltriplicate.

Cre Delivery Assay.

Uptake and delivery assays for Cre fusion proteins were performed aspreviously described (Cronican, J. J.; et al. ACS Chemical Biology 2010,5, 747). Briefly, proteins were diluted in serum-free DMEM and incubatedon the cells in 48-well plates for 4 h at 37° C. Following incubation,the cells were washed three times with PBS+20 U/mL heparin. The cellswere incubated a further 48 h in serum-containing media prior totrypsinization and analysis by flow cytometry. All flow cytometry werecarried out on a BD Fortessa flow cytometer (Becton-Dickinson) using530/30 nm and 610/20 nm filter sets. Toxicity for aurein 1.2 andcitropin 1.3 validation assays was determined using CELLTITER-GLO assay(Promega) in 96-well plates following manufacturer protocol. Toxicityfor alanine scan mutational analysis was determined with LIVE/DEADfixable far-red dead cell stain (Life Technologies) with 635 nm laserand 670/30 nm filter.

GR-m Cherry Translocation Assay.

One day prior to transfection 10,000 HeLa cells in 200 μL of DMEM (10%FBS, 1× Pen-Strep) were plated into single wells of a 96-well MATRICALglass bottom microplate (MGB096-1-2-LG-L) and allowed to adhereovernight. The following day, cells were transfected with GRmCherryusing LIPOFECTAMINE® 2000 technologies. Following transfection, cellswere allowed to recover overnight in DMEM (+10% FBS). The following day,cells were treated with dexamethasone (Dex) or 1 μM Dex-proteinconjugate in the presence or absence of inhibitor diluted into DMEM(without phenol red, +300 nM Hoescht33342). After 30 min, cells werewashed twice with 200 μL of HEPES-Krebs-Ringer's (HKR) buffer (140 mMNaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES at pH 7.4),after which 100 μL of HKR buffer was overlaid onto the cells and imageswere acquired on a Zeiss Axiovert 200M epifluorescence microscopeoutfitted with Ziess AxiocammRM camera and an EXFO-Excite series 120 Hgarc lamp. The translocation ratio (the ratio of median GFP intensity inthe nuclear and surrounding regions) for individual cells was measuredusing CELLPROFILER® as described 36. To examine the effect ofendocytosis inhibitors, HeLa cells were pretreated for 30 min with DMEM(without phenol red) containing inhibitors (80 μM Dynasore, 5 mM MBCD,50 μM EIPA, 200 nM bafilomycin or 200 nM wortmannin) at 37° C. for 30min before incubation with Dex or Dex-protein conjugates.

BirA Translocation Assay.

One day prior to transfection, 100,000 HeLa cells in 1 mL of DMEM (10%FBS, 1× PenStrep) were plated into single wells of a 12-well tissueculture plate and allowed to adhere overnight. Cells were transfectedwith mCherry-AP fusion protein using LIPOFECTAMINE® 2000 technologiesaccording to manufacture guidelines 24 h before protein treatment. Nextday, transfected cells were treated for 1 h at 37° C. with +36 GFP-BirAor aurein 1.2-+36 GFP-BirA diluted in serum-free DMEM at 250 nM, 500 nMand 1 μM concentrations. 250 nM+36 GFP-BirA+100 μM chloroquine was alsoused as a positive control for endosomal escape. The cells were washedthree times with PBS containing heparin to remove excess superchargedproteins that were not internalized. The cells were then treated with100 μL of 10 μM biotin and 1 mM ATP in PBS for 10 min. The reaction wasquenched with excess (10 μL of 8 mM) synthesized AP before cells weretrypsinized and lysed. To verify that extracellular BirA was notgenerating signal during lysis, 1 μM+36 GFP-BirA or aurein 1.2-+36GFP-BirA was added during the quench step to untreated wells. Cells werelysed with 100 μL of trypsin and lysed with QIASHREDDER columns(Qiagen). 30 μL of lysate was loaded onto 4-12% Bis-Tris Bolt gels inBolt-MES buffer (Life Technologies) and ran for 20 min at 200 volts.Gels were transferred to PVDF membrane using IBLOT2 transfer system(Life Technologies). Biotinylation was measured through western blottingusing the LI-COR quantitative infrared fluorescent antibodies and theOdyssey Imager detection system. To normalize for transfection and gelloading variables, the ratio of biotin signal to mCherry signal was usedfor comparison.

Cytosolic Fractionation Assay.

One day prior to fractionation, 4×10⁶ HeLa cells were plated in 20 mL ofDMEM (10% FBS, 1× PenStrep, no phenol red) in 175-cm² culture flasks andallowed to adhere for 15 hours. The following day, the media was removedfrom each flask and the cells were washed twice with clear DMEM (no FBS,no PenStrep, no phenol red). The media was replaced with 7 mL of clearDMEM containing +36 GFP or aurein 1.2-+36GFP at a concentration of 250nM, 500 nM, or 1 μM. Several flasks were treated with clear DMEM to beused as negative controls and to generate calibration curves with thecytosolic extracts. The cells were incubated for 30 min at 37° C., 5%CO₂ after which they were washed three times with PBS. Using acell-scraper, the cells were suspended in 5 mL of PBS, transferred intoa 15 mL Falcon tube, and pelleted at 500 g for 3 min. The cells wereresuspended in 1 mL PBS, counted using an automated cell counter (AutoT4, CELLOMETER®), and pelleted again at 500 g for 3 min. The cell pelletwas resuspended in ice-cold isotonic sucrose (290 mM sucrose, 10 mMimidazole, pH 7.0 with 1 mM DTT, and COMPLETE™, EDTA-free proteaseinhibitor cocktail) and transferred to a glass test tube on ice. Thecells were homogenized with an Omni TH homogenizer outfitted with astainless steel 5 mm probe for three 30 s pulses on ice with 30 s pausesbetween the pulses. The homogenized cell lysate was sedimented at 350 Kgin an ultracentrifuge (TL-100; Beckman Coulter) for 30 min at 4° C.using a TLA 120.2 rotor. The supernatant (cytosolic fraction) wasanalyzed in a 96-well plate on a fluorescence plate reader (SYNERGY 2,BioTek, excitation=485+/−10 nm, emission=528+/−10 nm). The concentrationof the protein conjugate in the cytosol was determined using a standardcurve relating fluorescence to known protein concentrations. To generatethe standard curve, known concentrations of +36 GFP and aurein 1.2-+36GFP between 0.2 nM and 1 μM were added to cytosolic extracts of theuntreated negative controls. For background subtraction, several wellscontaining cytosolic extracts from untreated cells were averaged, andthis average was subtracted from each well.

Total Protein Delivery Assay.

One day prior to the experiment, 100,000 HeLa cells/well were plated inDMEM (10% FBS, 1× PenStrep, no phenol red) in 6-well plates and allowedto adhere for 15 hours. The following day, the media was removed fromeach well and the cells were washed twice with clear DMEM (no FBS, noPenStrep, no phenol red). The media was replaced with 1 mL of clear DMEMcontaining +36 GFP or aurein 1.2-+36 GFP at concentrations of 250 nM,500 nM, or 1 μM. The cells were incubated for 30 min at 37° C., 5% CO₂after which they were washed three times with PBS containing 20 U/mLheparin (Sigma) to remove surface-bound cationic protein. The cells weretrypsinized for 5 min, pelleted in serum-containing DMEM for 3 min at500 g, washed with 1 mL PBS, and pelleted again for 3 min at 500 g. Thecell pellet was resuspended in 100 μL PBS. Flow cytometry was performedon a BD Accuri C6 Flow Cytometer at 25° C. Cells were analyzed in PBS(excitation laser=488 nm, emission filter=533+/−30 nm). At least 10,000cells were analyzed for each sample. For background subtraction, wellswere treated with clear DMEM only. The average of three untreated wellswas subtracted from each +36 GFP conjugate-containing well.

Microinjection of Proteins to Mouse Inner Ear.

P1-2 Gt(ROSA)26Sor^(tm14(CAG-tdTomato)Hze) mice were used for aurein1.2-+36-GFP-Cre and +36-GFP-Cre injection. The Rosa26-tdTomato mice werefrom the Jackson Laboratory. Animals were used under protocols approvedby the Massachusetts Eye &Ear Infirmary IACUC committee. Mice wereanesthetized by hypothermia on ice. Cochleostomies were performed bymaking an incision behind the ear to expose the cochlea. Glassmicropipettes held by a micromanipulator were used to deliver thecomplex into the scala media, which allows access to inner ear haircells. The total delivery volume for every injection was 0.4 μL percochlea and the release was controlled by a micromanipulator at thespeed of 69 nL/min.

Immunohistochemistry and Quantification.

5 days after injection, the mice were sacrificed and cochlea wereharvested by standard protocols (Sage, C.; et al. Science 2005, 307,1114). For immunohistochemistry, antibodies against hair-cell markers(Myo7a) and supporting cells (Sox2) were used following a previouslydescribed protocol (Sage C et al., supra). To quantify the number oftdTomato positive cells after aurein 1.2-+36-GFP-Cre and +36-GFP-Cre, wecounted the total number of inner and outer hair cells in a regionspanning 100 μm in the apex, middle, and base turn of the cochlea.

Results

Preparation of Antimicrobial Peptide (AMP) Conjugates of SuperchargedGFP-Cre Fusion Protein:

AMPs from the Antimicrobial Peptide Database (Wang, Z.; Wang, G. Nucleicacids research 2004, 32, D590) that are ≤25 amino acids long (tofacilitate their preparation and conjugation to +36 GFP), lackpost-translational modifications, and are not known to be toxic tomammalian cells. Based on these criteria, 36 AMPs were identifiedranging from 9 to 25 amino acids in length (Table 1). Each of thepeptides was synthesized on solid phase with an LPETGG (SEQ ID NO: 89)sequence appended to their C-terminus to enable sortase-catalyzedconjugation (Chen, I.; et al. Proceedings of the National Academy ofSciences 2011, 108, 11399) (FIG. 13B). Assembly of proteins usingsortase proved more amenable to rapid screening than the constructionand expression of the corresponding fusions, especially since severalAMP fusions do not express efficiently in E. coli.

The peptides were conjugated to purified GGG-(+36 GFP)-Cre using thepreviously described evolved sortase A enzyme (eSrtA) (Chen, I.; et al.Proceedings of the National Academy of Sciences 2011, 108, 11399).Sortase catalyzes the transpeptidation between a substrate containingthe C-terminal LPETGG (SEQ ID NO: 89) and a substrate containing anN-terminal glycine to form a native peptide bond linkage and a proteinidentical to the product of translational fusion.

TABLE 1 List of peptides chosen from the AntimicrobialPeptide Database (APD) APD Conjugation Label number Sequence efficiencyA AP00408 FLFPLITSFLSKVL 55% (SEQ ID NO: 36) B AP00405- FISAIASMLGKFL70% 11 (SEQ ID NO: 37) C AP00327 GWFDVVKHIASAV — (SEQ ID NO: 38) DAP01434 FFGSVLKLIPKIL — (SEQ ID NO: 39) E AP00013 GLFDIIKKIAESF 77%(SEQ ID NO: 34) F AP00025 HGVSGHGQHGVHG 20% (SEQ ID NO: 40) G AP00094FLPLIGRVLSGIL — (SEQ ID NO: 41) H AP00012 GLFDIIKKIAESI 28%(SEQ ID NO: 42) I AP00014 GLLDIVKKVVGAFGSL — (SEQ ID NO: 43) J AP00015GLFDIVKKVVGALGSL 13% (SEQ ID NO: 44) K AP00016 GLFDIVKKVVGAIGSL —(SEQ ID NO: 45) L AP00017 GLFDIVKKVVGTLAGL 18% (SEQ ID NO: 46) M AP00018GLFDIVKKVVGAFGSL — (SEQ ID NO: 47) N AP00019 GLFDIAKKVIGVIGSL —(SEQ ID NO: 48) O AP00020 GLFDIVKKIAGHIAGSI — (SEQ ID NO: 49) P AP00021GLFDIVKKIAGHIASSI — (SEQ ID NO: 50) Q AP00022 GLFDIVKKIAGHIVSSI(SEQ ID NO: 51) R AP00101 FVQWFSKFLGRIL 51% (SEQ ID NO: 52) S AP00351GLFDVIKKVASVIGGL 11% (SEQ ID NO: 53) T AP00352 GLFDIIKKVASVVGGL —(SEQ ID NO: 54) U AP00353 GLFDIIKKVASVIGGL  4% (SEQ ID NO: 35) V AP00567VWPLGLVICKALKIC  4% (SEQ ID NO: 55) W AP00597 NFLGTLVNLAKKIL 34%(SEQ ID NO: 56) X AP00818 FLPLIGKILGTIL 14% (SEQ ID NO: 57) Y AP00866FLPIIAKVLSGLL 86% (SEQ ID NO: 58) Z AP00870 FLPIVGKLLSGLL —(SEQ ID NO: 59) AA AP00875 FLSSIGKILGNLL 88% (SEQ ID NO: 60) AB AP00898FLSGIVGMLGKLF 70% (SEQ ID NO: 61) AC AP01211 TPFKLSLHL 81%(SEQ ID NO: 62) AD AP01249 GILDAIKAIAKAAG 20% (SEQ ID NO: 63) AEAP00013- LFDIIKKIAESF 63% G (SEQ ID NO: 64) AF AP00013-LFDIIKKIAESGFLFDIIKKIAESF — 2x (SEQ ID NO: 65) AG AP00722-GLLNGLALRLGKRALKKIIKRLCR — 75 (SEQ ID NO: 66) AH His13 GHHHHHHHHHHHHH —(SEQ ID NO: 67) AI AP00512 FKCRRWQWRM 42% (SEQ ID NO: 68) AJ AP00553KTCENLADTY — (SEQ ID NO: 69) Peptides were synthesized with a C-terminalLPETGG tag (SEQ ID NO: 89) to enable conjugation with an evolved sortase(eSrtA). Conjugation efficiencies were calculated based on LC-MS resultsusing peak abundance as determined through MaxEnt protein deconvolution.

The efficiency of eSrtA-mediated conjugation varied widely among thepeptides. Of the 36 peptides chosen for screening, 20 showed detectable(4% to 88%) sortase-mediated conjugation to +36 GFP-Cre, as observed byLC-MS, to generate desired peptide-LPETGGG-(+36 GFP)-Cre fusion proteins(“LPETGGG” disclosed as SEQ ID NO: 96) (Table 1). Unreacted peptide wasremoved by ultrafiltration with a 30-kD molecular weight cut offmembrane.

Primary Screen for Endosomal Escape.

The ability of each peptide-(+36 GFP)-Cre recombinase fusion was assayedwhen added to culture media to effect recombination in BSR.LNL.tdTomatocells (Cronican, J. J.; et al. ACS Chemical Biology 2010, 5, 747), ahamster kidney cell line derived from BHK-21. Because Cre recombinasemust enter the cell, escape endosomes, enter the nucleus, and catalyzerecombination to generate tdTomato fluorescence, this assay reflects theavailability of active, non-endosomal recombinase enzyme that reachesthe nucleus. As a positive control, cells were treated with +36 GFP-Creand chloroquine, a known endosome-disrupting small molecule (Dijkstra,J.; et al. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research1984, 804, 58).

The reporter BSR.LNL.tdTomato cells were incubated with 250 nM of eachpeptide-(+36 GFP)-Cre protein in serum-free media. In the absence of anyconjugated peptide, treatment of reporter cells with 250 nM+36 GFP-Creprotein resulted in 4.5% of the cells expressing tdTomato, consistentwith previous reports 18. The same concentration of protein incubatedwith 100 μM chloroquine as a positive control resulted in an average of48% recombined cells (FIG. 14). The results of chloroquine treatmentvaried substantially between independent replicates. As chloroquine isknown to be toxic to cells above 100 μM, it was speculated that thisvariability arises from the small differences between chloroquine'sefficacious and toxic dosages.

Ten of the screened peptide conjugates resulted in recombinationefficiencies that were significantly above that of +36 GFP-Cre (FIG.14). The most potent functional delivery of Cre was observed with aurein1.2-+36 GFP-Cre (Table 1, entry E). Treatment with aurein 1.2-+36GFP-Cre resulted in an average of 40% recombined cells, comparable tothat of the chloroquine positive control (FIG. 14). To investigate theimpact of differential conjugation efficiency on peptide performance,citropin 1.3 (Table 1, entry U), which displayed a moderate level ofrecombination and the lowest level of conjugation (4%), was compared toaurein 1.2, which has the highest level of recombination and also a highlevel of conjugation (77%).

Both aurein 1.2-+36 GFP-Cre and citropin 1.3-+36 GFP-Cre were cloned,expressed, and purified as fusion proteins. The recombination signalfrom treatment with 250 nM of expressed aurein 1.2-+36 GFP-Cre was10.4-fold above that of +36 GFP-Cre. In contrast, treatment with 250 nMexpressed citropin 1.3-+36 GFP-Cre did not induce any enhanced Credelivery. When the treatment concentration was increased to 1 μM, aurein1.2-+36 GFP-Cre and citropin 1.3-+36 GFP-Cre resulted in 3.8-fold and3.0-fold higher recombination levels, respectively, than that of +36GFP-Cre alone (FIG. 15A). These results evidence that while aurein 1.2and citropin 1.3 both enhance the delivery of functional, non-endosomal+36 GFP-Cre protein at high concentrations, aurein 1.2 has greaterefficacy than citropin 1.3 at lower concentrations.

Next, the toxicity of each fusion protein was evaluated at a range ofconcentrations (125 nM to 1 μM) using an ATP-dependent cell viabilityassay at 48 h after treatment. For +36 GFP-Cre, it was observed thatthere was no cellular toxicity up to 1 μM treatment, which resulted in85% viable cells. Cells treated with 250 nM recombinant aurein 1.2-+36GFP-Cre and citropin 1.3-+36 GFP-Cre displayed 87% and 84% viability,respectively. Applying 1 μM treatments decreased cell viability to 70%and 66%, respectively (FIG. 15B). In light of its activity and lowcytotoxicity at 250 nM, the ability of aurein 1.2 to enhance cytosolicprotein delivery was characterized in depth.

Site-Directed Mutagenesis of Aurein 1.2.

Aurein 1.2 (GLFDIIKKIAESF; SEQ ID NO: 34) is a potent AMP excreted fromthe Australian tree frog, Litoria aurea (Rozek, T.; et al. RapidCommunications in Mass Spectrometry 2000, 14, 2002). Interestingly,citropin 1.3 (GLFDIIKKVASVIGGL; SEQ ID NO: 35) is a closely relatedpeptide and is excreted from a different Australian tree frog, Litoriacitropa (Wegener, K. L.; et al. European journal of biochemistry/FEBS1999, 265, 627). While the properties of aurein 1.2 have beeninvestigated for its anti-bacterial and anti-tumorigenic abilities((Rozek, T.; et al. Rapid Communications in Mass Spectrometry 2000, 14,2002), its ability to enhance endosomal escape or macromolecule deliveryhas not been previously reported. The free peptide is thought to adoptan amphipathic alpha helical structure in solution, but the length ofthe helix is too short to span a lipid bilayer (Balla, M.; Bowie, J. H.;Separovic, F. European Biophysics Journal 2004, 33, 109. Therefore itwas theorized that aurein 1.2 disrupts membranes through a “carpetmechanism” in which peptides bind to the membrane surface in a mannerthat allows hydrophobic residues to interact with lipid tails andhydrophilic residues to interact with polar lipid head groups(Fernandez, D. I.; et al. Physical Chemistry Chemical Physics 2012, 14,15739). Above a critical concentration, the peptides are thought toalter the curvature of the membrane enough to break apart thecompartment.

TABLE 2 Site-directed mutagenesis of aurein 1.2 Label SequenceAurein 1.2 GLFDIIKKIAESF (SEQ ID NO: 34) G1AALFDIIKKIAESF (SEQ ID NO: 70) L2A GAFDIIKKIAESF (SEQ ID NO: 71) F3AGLADIIKKIAESF (SEQ ID NO: 72) D4A GLFAIIKKIAESF (SEQ ID NO: 73) I5AGLFDAIKKIAESF (SEQ ID NO: 74) I6A GLFDIAKKIAESF (SEQ ID NO: 75) K7AGLFDIIAKIAESF (SEQ ID NO: 76) K8A GLFDIIKAIAESF (SEQ ID NO: 77) I9AGLFDIIKKAAESF (SEQ ID NO: 78) E11A GLFDIIKKIAASF (SEQ ID NO: 79) S12AGLFDIIKKIAEAF (SEQ ID NO: 80) F13A GLFDIIKKIAESA (SEQ ID NO: 81) K7HGLFDIIHKIAESF (SEQ ID NO: 82) K8H GLFDIIKHIAESF (SEQ ID NO: 83) E11HGLFDIIKKIAHSF (SEQ ID NO: 84) K7R GLFDIIRKIAESF (SEQ ID NO: 85) K8RGLFDIIKRIAESF (SEQ ID NO: 86) E11R GLFDIIKKIARSF (SEQ ID NO: 87) E11DGLFDIIKKIADSF (SEQ ID NO: 88) An alanine scan was performed on aurein1.2 to determine positions that tolerate mutation. Charged amino acidsat tolerant positions were then replaced with histidines or othercharged amino acids in an attempt to increase endosomal escapeefficiency. All constructs were expressed as recombinant fusion proteinswith +36 GFP-Cre.

To identify the residues involved in enhancing non-endosomal proteindelivery, an alanine scan of the 13 amino acid positions of aurein 1.2was performed, by cloning, expressing, and purifying each alanine mutantof aurein 1.2-+36 GFP-Cre. The resulting fusion proteins were assayed inBSR.LNL.tdTomato reporter cells as described above (Table 2). Sevenpositions were moderately to highly intolerant of alanine substitution.Six positions retained >70% of the activity of unmutated aurein 1.2-+36GFP-Cre (FIG. 16A). At each of these tolerant positions, which includedthree positions with charged residues (K7, K8, and E11 from Table 2),additional mutations were generated in an effort to improve activity. Intotal, 19 mutants of aurein 1.2 were generated and tested using the Crerecombination assay. Two of the aurein variants, K8R and S12A, exhibitedpotentially improved overall recombination efficiency but also increasedtoxicity at 250 nM (FIG. 16B). Given this increase in toxicity, it wasdecided to focus on the original peptide, aurein 1.2, and proceeded tocharacterize its potency through a series of complementary secondaryassays.

Independent Assays of Endosomal Escape.

Although endosomal escape is widely considered to be the majorbottleneck of cationic protein delivery (Sahay, G.; et al. NatureBiotechnology 2013, 31, 653), few assays quantify the ability ofproteins to escape endosomes on a single-cell basis. To quantifycytosolic delivery of supercharged proteins in individual cells, aglucocorticoid receptor (GR) translocationassay (Yu, P.; Liu, B.;Kodadek, T. Nat Biotech 2005, 23, 746) was applied. In untreated HeLacells expressing mCherry-labeled GR (GR-mCherry), the GR distributesnearly uniformly throughout the cell interior, resulting in anuclear-to-cytoplasm translocation ratio (TR) of 1.17 (FIGS. 17A and17B). Upon treatment with the cell-permeable glucocorticoiddexamethasone-21-thiopropionic acid (S^(Dex)) at a concentration of 1 μMfor 30 min, GR-mCherry relocates almost exclusively to the nucleus,resulting in a TR of 3.77 (FIGS. 17A and 17B).

Dexamethasone conjugates of +36 GFP (+36 GFP^(Dex)) and aurein 1.2-+36GFP (aurein 1.2-+36 GFP^(Dex)) were generated via sortase-mediatedconjugation. Conjugated to these proteins, S^(Dex) is no longer cellpermeable and cannot activate the GR for nuclear translocation unlessthe protein-S^(Dex) conjugate can access the cytosol. Treatment of HeLacells expressing GR-mCherry with 1 μM aurein 1.2-+36 GFP^(Dex) for 30min resulted in a TR of 2.62, which was significantly greater (p<0.05)than that of +36 GFP^(Dex) (TR=2.23). As positive controls, these cellswere treated with canonical cell permeable peptides (Tat^(Dex) andArg8^(Dex) (SEQ ID NO: 102)) and miniature proteins containing apenta-Arg motif that reach the cytosol intact, with efficienciesexceeding 50% (5.3^(Dex) and ZF 5.3^(Dex)) (LaRochelle, J. R.; et al.Journal of the American Chemical Society 2015, 137, 2536). Aurein1.2-+36 GFP^(Dex) (TR=2.62), activated significantly greater levels ofGR-mCherry translocation (p<0.001) than Tat^(Dex) (TR=1.87) andArg8^(Dex) (SEQ ID NO: 102) (TR=1.63) and similar levels evoked byminiature proteins 5.3^(Dex) (TR=2.62) and ZF 5.3^(Dex) (TR=2.38) (FIGS.17A and 17B). Taken together, these results evidence that aurein 1.2-+36GFP^(Dex) exhibits an improved ability to access the cytoplasm over +36GFP^(Dex) and canonical cell permeable peptides.

As an additional, independent assay of non-endosomal protein delivery,the ability of aurein 1.2 to enhance the nonendosomal delivery of anevolved biotin ligase (BirA) enzyme was tested (Howarth, M.; Ting, A. Y.Nature protocols 2008, 3, 534). BirA catalyzes the biotinylation of a15-amino acid acceptor peptide (AP). A mCherry-AP fusion plasmid wastransfected into HeLa cells. Biotinylation of mCherry can only occur inthe presence of cytosolic BirA. To assess the non-endosomal delivery of+36 GFP-BirA protein, mCherry-AP biotinylation was quantified by Westernblot using fluorophore-labeled streptavidin and normalized to themCherry signal. Treatment with 250 nM aurein 1.2-+36 GFP-BirA resultedin a 50% increase in biotinylation signal compared with 250 nM of +36GFP-BirA alone. A dose-dependent increase in AP-biotinylation acrosstreatment concentrations (250 nM, 500 nM, and 1 μM) for both aurein1.2-(+36GFP)-BirA and unfused +36 GFP-BirA constructs was observed.These results are consistent with the results of the GR translocationassay, and further evidence that aurein 1.2 enhances the endosomalescape of superpositively charged proteins.

In order to directly quantify the increase in non-endosomal deliveryresulting from aurein 1.2, a cytosolic fractionation experiment wasperformed to calculate the cytosolic concentrations of deliveredprotein. HeLa cells were treated with +36 GFP or aurein 1.2-+36 GFP at250 nM, 500 nM, and 1 μM. After 30 min of treatment, cells were washed,homogenized, and fractionated by ultracentrifugation. The cytosolicconcentration of delivered protein was calculated from the GFPfluorescence of the cytosolic fraction together with a standard curverelating fluorescence to known concentrations of +36 GFP and aurein1.2-+36 GFP added to cytosolic extract. At 250 nM, treatment with aurein1.2-+36 GFP resulted in ˜5-fold more delivered cytosolic protein thantreatment with +36 GFP alone. This difference decreased with increasingprotein concentration, likely due to the influence of alternate uptakepathways or delivery bottlenecks at high protein concentrations. Incontrast, the total amount of aurein 1.2-+36GFP versus +36 GFP taken upby cells was similar, with aurein 1.2-+36 GFP showing 1.3-fold highertotal cellular uptake at 250 nM. These results directly demonstrate thataurein 1.2 increases the cytosolic concentration of cationic proteinsthat enter cells predominantly through endosomes (McNaughton, B. R.; etal. Proceedings of the National Academy of Sciences 2009, 106, 6111;Thompson, David B.; et al. Chemistry & Biology 2012, 19, 831) and areconsistent with the above findings that aurein 1.2 has the greatesteffect on enhancing non-endosomal delivery at ˜250 nM (FIG. 15A).

Effect of Endocytic Inhibitors on +36 GFP and Aurein 1.2-+36 GFPDelivery.

Endocytosis plays a key role in the cytosolic delivery ofsuperpositively charged proteins (Thompson, David B.; et al. Chemistry &Biology 2012, 19, 831). To probe the role of endocytosis in the deliveryof supercharged proteins with or without aurein 1.2, cells expressingGR-mCherry were treated with either +36 GFP^(Dex) or aurein1.2-+36GFP^(Dex) in the presence of known endocytic inhibitors. Thecortical actin remodeling inhibitor N-ethyl-isopropyl amiloride (EIPA),the cholesterol-sequestering agent methyl-β-cyclodextrin (MBCD), and theendosomal vesicular ATPase inhibitor bafilomycin (Baf) all stronglyreduced the ability of both proteins to stimulate GRmCherrytranslocation. Blocking maturation of Rab5⁺ vesicles by treatment withthe phosphatidylinositol 3-kinase inhibitor wortmannin (Wort) did notinfluence reporter translocation of either +36 GFP^(Dex) or aurein1.2-+36 GFP^(Dex) (FIGS. 17C and 17D). In contrast, treatment with thesmall-molecule dynamin II inhibitor Dynasore (Dyna) significantlysuppressed the ability of +36 GFP^(Dex) to stimulate GR-mCherrytranslocation (TR=1.64) (FIG. 17C) but had little influence on thecytosolic delivery of aurein 1.2-+36GFP^(Dex) (TR=2.30) (FIG. 17D).Taken together, these results evidence that active endocytosis isrequired for uptake of +36 GFP and aurein 1.2-+36GFP into the cellinterior, and that the two proteins may traffic differently into thecell interior.

Aurein 1.2 can Greatly Increase Protein Delivery Efficiency In Vivo.

To evaluate the ability of aurein 1.2 to increase the efficacy ofcationic protein delivery in vivo, proteins were delivered to the innerear of Cre reporter transgenic mice that express tdTomato uponCre-mediated recombination. This animal model was chosen due to itsconfined injection volume, the presence of well-characterized celltypes, and the existence of genetic deafness models that wouldfacilitate future studies of protein delivery to treat hearing loss. +36GFP-Cre alone can be delivered to mouse retina (Cronican, J. J.; et al.ACS Chemical Biology 2010, 5, 747), albeit resulting in only modestlevels of recombination consistent with inefficient endosomal escape.

Anesthetized postnatal day 2 (P2) mice were injected with 0.4 μL of 50μM+36 GFP-Cre or aurein 1.2-+36 GFP-Cre solutions in the scala media toaccess the cochlear cells. Five days after injection, the cochleas wereharvested for immunolabeling of inner ear cell markers and imaging fortdTomato florescence (FIG. 18A). Both the hair cells (Myo7a+) andsupporting cells (Myo7a−) were evaluated for td Tomato signal. The totalnumber of hair cells and supporting cells (by DAPI labeling) in thesensory epithelium (SE) was used to determine the relative toxicity ofaurein 1.2-+36 GFP-Cre to the baseline treatment of +36 GFP-Cre (FIG.18A). Overall, an average of 96%, 92% and 66% of cochlear cells survivedaurein 1.2-+36 GFP-Cre treatment as compared to +36 GFP-Cre treatment inthe apex, middle, and base tissue samples, respectively (FIG. 18A). +36GFP-Cre treatment resulted in low levels of recombination only in innerhair cells (IHC) of the apex of the cochlea (4.4%) but not in the middleor base of the cochlear hair cells or any cochlear supporting cells. Incontrast, treatment with aurein 1.2-+36 GFP-Cre resulted in very highCre-mediated recombination levels throughout the apex, middle, and basesamples of outer hair cells (OHC) (96%, 91%, and 69%, respectively),inner hair cells (100%, 94%, and 70%, respectively), as well assupporting cells (arrows) (FIGS. 18A and 18C).

The observed levels of recombination in the inner hair cells from aurein1.2-+36 GFP-Cre are comparable to that of adeno-associated virus type 1(AAV1) gene transfection (Akil, O.; et al. Neuron 2012, 75, 283). Forouter hair cells, it similar levels of recombination was previouslyshown using liposome-mediated delivery of supernegatively-chargedGFP-Cre (Zuris, J. A.; et al. Nat Biotech 2015, 33, 73). The aurein1.2-+36 GFP-Cre delivery system is the only method that showedsignificant recombination levels in both inner and outer hair cells(Akil, O.; et al. Neuron 2012, 75, 283; Taura, A.; et al. Neuroscience2010, 166, 1185), and does not require any virus or other moleculesbeyond a single polypeptide. Significantly, aurein 1.2-+36 GFP-Cre alsoextended delivered recombinase activity to additional cochlearsupporting cells. These results evidence that the aurein 1.2-+36 GFP-Credelivery system to be a promising method for in vivo protein deliveryinto both hair cells and supporting cells of the inner ear (Izumikawa,M.; et al. Nature Medicine 2005, 11, 271).

DISCUSSION

A 13-residue peptide, aurein 1.2, was discovered that can increase theefficiency of non-endosomal protein delivery by screening a panel ofknown membrane-active peptides. The results from a small screen of 22peptides are consistent with our hypothesis that some peptides canselectively disrupt the endosomal membrane without disrupting themammalian cell membrane. The effectiveness of aurein 1.2 is highlydependent on its sequence, as several other closely related peptides didnot enhance protein delivery (Tables 1 and 2). Subtle differences inamino acid composition led to dramatic changes in membrane activityamong peptides tested, highlighting the difficulty of rationallydesigning peptides that enhance non-endosomal delivery. Moreover, thelack of correspondence between peptide cationic charge and non-endosomaldelivery efficiency evidences that aurein 1.2 does not enhancenon-endosomal delivery simply by promoting endocytosis. While none ofthe tested variants of aurein 1.2 substantially outperformed theoriginal peptide, several amino acids were identified that could bealtered without loss of activity. These findings also provide a startingpoint for further optimization to discover next-generation endosomolyticpeptides with improved efficacy and reduced toxicity.

Four independent assays for non-endosomal protein delivery (Crerecombination, GR translocation, BirA activity on a cytoplasmic peptide,and cytosolic fractionation), together with the peptide mutationalstudies described above, collectively evidence that aurein 1.2-fusionenhances endosomal escape of superpositively charged proteins. Moreover,these assays collectively demonstrated the ability of aurein 1.2 tomediate the non-endosomal delivery of +36 GFP fused to differentproteins (or small molecules), evidences that aurein 1.2 facilitiesendosomal escape.

The in vivo protein delivery experiments described above revealeddramatic increases in non-endosomal functional Cre recombinase deliveryinto the diverse inner ear cell types including hair cells andsupporting cells of live mice upon fusion with aurein 1.2. Indeed,aurein 1.2-fused +36 GFP-Cre construct resulted in highly efficientrecombination levels across the main cochlear sensory epithelial cellclasses studied in this work, all but one of which were unaffected by+36 GFP-Cre treatment. Taken together, these results evidence thataurein 1.2 is a 13-residue, potent, genetically encodable, endosomeescape-enhancing peptide that can greatly increase the efficiency ofnon-endosomal cationic protein delivery in vitro and in vivo withoutrequiring the use of additional components beyond the protein ofinterest.

What is claimed is:
 1. A method of treating deafness, associated with anautosomal dominant genetic mutation in a TMC1 gene in a patient in needthereof, comprising: administering to an inner ear of the patient atherapeutically effective amount of a chimeric molecule consisting of amembrane destabilizing domain, a gene editing agent, wherein the geneediting agent is Cas9, and a gRNA targeting a TMC1 gene, therebytreating deafness.
 2. The method of claim 1, wherein the chimericmolecule targets one or more genetic loci associated with deafness in apatient and modulates replication, expression, function or activity ofthe genetic locus in hair cells, stem cells, or supporting cells.
 3. Themethod of claim 2, wherein the one or more genetic loci comprisechromosomal abnormalities having deletions.
 4. The method of claim 1,wherein the genetic mutations further comprise one or more monogenicmutations.
 5. The method of claim 1, wherein the chimeric moleculefurther comprises a supercharged protein or variants thereof.
 6. Themethod of claim 5, wherein the supercharged protein is green fluorescentprotein (s-GFP), or variants thereof.
 7. The method of claim 1, whereinthe gene editing agents comprise: transcriptional activators,transcriptional repressors, recombinases, nucleases, DNA-bindingproteins or nucleic acids, or combinations thereof.
 8. The method ofclaim 1, wherein the chimeric molecule further comprises antimicrobialor membrane destabilizing peptides linked to the s-GFP, or variantsthereof.
 9. The method of claim 1, wherein the chimeric molecule isencapsulated in a cationic lipid formulation.
 10. The method of claim 1,wherein the chimeric molecule comprises any one or more sequences havinga sequence identity of at least about 75% to SEQ ID NO:
 95. 11. Themethod of claim 10, wherein the one or more sequences comprise SEQ IDNO: 95.